Histidyl-trna synthetase-fc conjugates

ABSTRACT

The present invention provides histidyl-tRNA synthetase and Fc region conjugate polypeptides (HRS-Fc conjugates), such as HRS-Fc fusion polypeptides, compositions comprising the same, and methods of using such conjugates and compositions for treating or diagnosing a variety of conditions. The HRS-Fc conjugates of the invention have improved controlled release properties, stability, half-life, and other pharmacokinetic and biological properties relative to corresponding, unmodified HRS polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/577,992, filed Sep. 20, 2019, which is a Continuation of U.S.application Ser. No. 16/122,231, filed Sep. 5, 2018; which is aContinuation of U.S. application Ser. No. 15/415,369, filed Jan. 25,2017, now U.S. Pat. No. 10,093,915, issued on Oct. 9, 2018; which is aContinuation of U.S. application Ser. No. 14/214,491, filed Mar. 14,2014, now U.S. Pat. No. 9,587,235, issued on Mar. 7, 2017; which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.61/789,011, filed Mar. 15, 2013, each of which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR_116_05 US_ST25.txt. The text file is about400 KB, was created on Apr. 7, 2020, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

The present invention relates generally to conjugates, such as fusionpolypeptides, of one or more histidyl-tRNA synthetase (HRS)polypeptide(s) and immunoglobulin Fc region(s), compositions comprisingthe same, and methods of using such polypeptides and compositions fortreating or diagnosing a variety of conditions.

Description of the Related Art

Physiocrines are generally small, naturally-occurring protein domainsfound in the aminoacyl-tRNA synthetases (AARSs) gene family of higherorganisms, which are not required for the well-established role ofaminoacyl-tRNA synthetases in protein synthesis. Until the Physiocrineparadigm was discovered, aminoacyl-tRNA synthetases, a family of about20 enzymes, were known only for their ubiquitous expression in allliving cells, and their essential role in the process of proteinsynthesis. More recent scientific findings however now suggest thataminoacyl-tRNA synthetases possess additional roles beyond proteinsynthesis and in fact have evolved in multicellular organisms to playimportant homeostatic roles in tissue physiology and disease.

Evidence for the existence of the non-canonical function of AARSsincludes well defined sequence comparisons that establish that duringthe evolution from simple unicellular organisms to more complex lifeforms, AARSs have evolved to be more structurally complex through theaddition of appended domains, without losing the ability to facilitateprotein synthesis.

Consistent with this hypothesis, a rich and diverse set of expandedfunctions for AARSs have been found in higher eukaryotes, and inparticular for human tRNA synthetases. This data, which is based both onthe direct analysis of individual domains, as well as the discovery ofmutations in genes for tRNA synthetases that are causally linked todisease, but do not affect aminoacylation or protein synthesis activity,suggests that these newly appended domains, or Physiocrines, are centralto the newly acquired non-canonical functions of AARSs.

Additionally, there is increasing recognition that specific tRNAsynthetases such as histidyl-tRNA synthetase (HRS) can be released orsecreted from living cells and can provide important locally actingsignals with immunomodulatory, chemotactic, and angiogenic properties.Direct confirmation of the role of AARS as extracellular signalingmolecules has been obtained through studies showing the secretion andextracellular release of specific tRNA synthetases, as well as thedirect demonstration that the addition of fragments of the tRNAsynthetases comprising the newly appended domains (Physiocrines), butnot other fragments lacking these domains, are active in a range ofextracellular signaling pathways. These Physiocrines such as HRSrepresent a new and previously untapped opportunity to develop new firstin class therapeutic proteins to treat human disease.

To best exploit these and other activities in therapeutic or diagnosticsettings, there is a need in the art for HRS polypeptides havingimproved pharmacokinetic properties. These improved therapeutic forms ofthe HRS polypeptides enable the development of more effectivetherapeutic regimens for the treatment of various diseases anddisorders, and require significantly less frequent administration thanthe unmodified proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structural make-up of an exemplaryimmunoglobulin, and provides an overview of antibody classes andsubclasses.

FIG. 2 shows an alignment of Fc regions from human IgA1 (SEQ ID NO:156),IgA2 (SEQ ID NO:157), IgM (SEQ ID NO:158), IgG1 (SEQ ID NO:159), IgG2(SEQ ID NO:160), IgG3 (SEQ ID NO:161), IgG4 (SEQ ID NO:162), and IgE(SEQ ID NO:163). The secondary structure of Fca is shown above thesequences. Carets (A) and asterisks (*) show residues that contributerespectively to 0-4% and 5-12% of the binding surface.

FIG. 3 shows the results of SDS-PAGE analysis under reducing and nonreducing conditions of full length HRS and HRS(1-506). The results showthat HRS(1-506) dramatically reduces the formation of disulfide mediatedinterchain bond formation compared to the full length protein. Samples(10 μg) were loaded on a 4-12% Bis-Tris gel, using a MOPS-SDS runningbuffer.

FIG. 4 shows the anti-inflammatory properties of an exemplaryHRS-derived polypeptide in a TNBS-induced mouse model of colitis.Studies were performed on male BDF-1 mice, with 12 mice/group. TNBS andbudesonide were added at 5 mg/kg to the water. HRS(1-60) (Resokine,(HisRS^(N4))) was administered daily by IV injection, starting 3 daysprior to TNBS treatment, at a concentration of 1 or 5 mg/kg. This figureshows the percent (%) survival of treated and untreated mice over about80 hours.

FIGS. 5A-5B show the dosing regimen used to evaluate the therapeuticutility of HRS(1-506) in the statin myopathy model. FIG. 5A shows thetreatment dosing groups which included vehicle (n=11), 0.3 mpkHRS(1-506) (n=8), 1.0 mpk HRS(1-506) (n=8), 3.0 mpk HRS(1-506) (n=8);FIG. 5B shows the results of Troponin C measurements after 15 days oftreatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg. Thefigure shows the positive effect of HRS(1-506) in reducing statininduced troponin C induction.

FIG. 6A shows the results of CK measurements after 12 days of treatmentwith statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg; FIG. 6B shows thesame data after 15 days of treatment. The figure shows the positiveeffect of HRS(1-506) in reducing statin induced CK levels.

FIG. 7 shows the levels of circulating HARS after 15 days of treatmentwith statins compared to the vehicle control. The figure shows thatstains induce the release of extracellular HARS.

FIG. 8 shows representative H&E images of hamstring sections at 10×magnification after 15 days of treatment with statins+/−HRS(1-506) at0.3, 1.0, and 3.0 mg/Kg.

FIG. 9 shows the results of gene expression profiling of statin treatedrat hamstrings. The data depicts changes in the expression of 137 genesselected to track markers of muscle, and immune cell function,inflammation, metabolic status, tissue recovery, muscle growth andatrophy. Gene expression values were normalized to reference genes andrepresented as fold change vs. the vehicle treated group.

FIGS. 10A-10B show the results of gene expression profiling of statintreated rat hamstrings. The data in FIG. 10A depicts changes in theexpression of 137 genes (as in FIG. 7) to compare the relative changesin gene expression of statin treated animals compared to vehicle treatedanimals. FIG. 10B shows the relative changes in gene expression ofstatin treated animals that were also treated with HRS(1-506) comparedto animals treated with statin alone.

FIG. 11 shows the results of gene expression profiling of statin treatedrat hamstrings of 10 diabetes/metabolic syndrome related genes after 15days of treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIG. 12 shows the results of gene expression profiling of statin treatedrat hamstrings of 26 immune cell marker genes after 15 days of treatmentwith statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIGS. 13A-13B and FIGS. 13C-13D show the results of gene expressionprofiling of the CD11a, CD11b, CD8a, and CD8b genes in statin treatedrat hamstrings after 15 days of treatment with statins+/−HRS(1-506) at0.3, 1.0, and 3.0 mg/Kg.

FIGS. 14A-14B and FIG. 14C show the results of gene expression profilingof the CD18, CCR5 and CD45R genes in statin treated rat hamstrings after15 days of treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0mg/Kg.

FIG. 15 shows the results of gene expression profiling of 17inflammatory marker genes in statin treated rat hamstrings after 15 daysof treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIGS. 16A-16B and FIGS. 16C-16D show the results of gene expressionprofiling of the inflammatory cytokines IL-6, MCP1, IL-10, andinterferon-gamma (IFN-γ) in statin treated rat hamstrings after 15 daysof treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIG. 17 shows the results of gene expression profiling of statin treatedrat hamstrings of 14 adhesion, development, and fibrosis related genesafter 15 days of treatment with statins+/−HRS(1-506) at 0.3, 1.0, and3.0 mg/Kg.

FIG. 18 shows the results of gene expression profiling of statin treatedrat hamstrings of 14 muscle wasting/atrophy related genes after 15 daysof treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIG. 19A shows the results of gene expression profiling of statintreated rat hamstrings of 14 muscle wasting/atrophy related genes after15 days of treatment with statins+/−HRS(1-506) at 0.3, 1.0, and 3.0mg/Kg. FIG. 19B shows specific changes in MMP-3, and FIG. 19C showsspecific changes in MMP-9 gene expression under the same conditions.

FIG. 20 shows the results of gene expression profiling of statin treatedrat hamstrings of 29 myogenesis related genes after 15 days of treatmentwith Statins+/−HRS(1-506) at 0.3, 1.0, and 3.0 mg/Kg.

FIG. 21 shows the results of SDS-PAGE analysis of purified Fc fusionproteins. Lane 1: See Blue Plus2 protein ladder (Life Technologies).Lane 2 and 6: Fc-HRS(2-60) lot#472. Lane 3 and 7: HRS(1-60)-Fc lot#473.Lane 4 and 8: Fc-HRS(2-60) lot#480. Lane 5 and 9: HRS(1-60)-Fc lot#482.Lanes 2-5 were run under non-reduced conditions, and lanes 6-9 reducedconditions.

FIG. 22 shows an analytical size-exclusion HPLC analysis ofrepresentative purified Fc-HRS(2-60) fusion after Protein A, cationexchange, and hydroxyapatite chromatography (overlay of duplicateinjections). Purity is 99.2% main peak, and 0.8% high molecular weight(HMW) species.

FIG. 23A shows the time versus concentration of HRS(1-60) followingeither intravenous or subcutaneous injection to mice. FIG. 23B shows thetime versus concentration of Fc-HRS(2-60) and HRS(1-60)-Fc followingintravenous injection to mice. FIG. 23C shows the time versusconcentration of Fc-HRS(2-60) and HRS(1-60)-Fc following subcutaneousinjection to mice.

FIG. 24A shows disease activity index (DAI) scores at termination inmice treated with different HRS-Fc fusion proteins. Bars represent themean DAI (+SEM) for each treatment group. The DAI incorporatesinformation on bleeding and diarrhea together with a score for weightloss. FIG. 24B shows colon weight: length ratio at termination in micetreated with compounds. Bars represent the mean ratio (±SEM) for eachtreatment group.

FIG. 25 shows an overview of transcriptional changes in TNBS study.Relative transcriptional changes in TNBS treated animals (group 2),animals treated with TNBS and budesonide (group 3), TNBS and testarticle A (HRS(1-60); group 4), and TNBS and test article B(Fc-HRS(2-60); groups 5 and 6) are shown following normalization tonaïve animals (group 1). Each dot in the scatter plot represents a genemeasured. 7 genes in group 2 were up-regulated more than 10-fold (IL6,IL1b, MCP-1, MMP3, MMP9, CD11b, and IL10).

FIGS. 26A-26C and FIGS. 26D-26F and FIG. 26H show the immune andinflammatory related genes up regulated by TNBS. Relativetranscriptional changes of individual genes in TNBS treated animals(group 2), animals treated with TNBS and budesonide (group3), TNBS andtest article A (HRS(1-60); group 4), and TNBS and test article B(Fc-HRS(2-60); groups 5 and 6) are shown following normalization tonaïve animals (group 1). Each dot in the scatter plot represents theabundance of the gene of interest in each animal within the group.Significance was calculated using a student's t-test where *=p-value<0.05 and **=p-value <0.01.

FIGS. 27A-27D shows the relative percentages of different T cellpopulations in the spleens of naïve mice or mice treatedintracolonically with TNBS to induce experimental colitis, treated withTNBS±0.5 mg/kg Fc-HRS(2-60). Shown are the percentage of livelymphocytes stained for (27A) CD3, (27B) CD8, (27C) CD4, and (27D) CD25and FoxP3. Treg cells were additionally gated on CD4⁺ cells.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate generally to histidyl-tRNAsynthetase (HRS) polypeptide conjugates having one or moreimmunoglobulin Fc regions covalently attached thereto, pharmaceuticalcompositions comprising such molecules, methods of manufacture, andmethods for their therapeutic use. Among other advantages, the HRS-Fcconjugates of the present invention can possess improved pharmacokineticproperties and/or improved therapeutically relevant biologicalactivities, relative to corresponding, un-modified HRS polypeptides.

Certain embodiments therefore include HRS fusion polypeptides,comprising a HRS polypeptide that comprises an amino acid sequence atleast 80% identical to any one of SEQ ID NOS:1-106, 170-181, or 185-191or a sequence of any of Tables D1, D3-D6, or D8, and at least one Fcregion fused to the C-terminus, the N-terminus, or both of the HRSpolypeptide. In some embodiments, the HRS polypeptide comprises,consists, or consists essentially of an amino acid sequence at least 90%identical to any of SEQ ID NOS:1-106, 170-181, or 185-191 or a sequenceof any of Tables D1, D3-D6, or D8. In particular embodiments, the HRSpolypeptide comprises, consists, or consists essentially of an aminoacid sequence of any one of SEQ ID NOS:1-106, 170-181, or 185-191 or asequence of any of Tables D1, D3-D6, or D8.

In particular embodiments, the HRS polypeptide comprises amino acidresidues 2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO:1, oran amino acid sequence at least 90% identical to residues 2-40, 2-45,2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO: 1. In some embodiments,the HRS polypeptide is up to about 40-80 amino acids in length andcomprises residues 2-45 of SEQ ID NO:1. In specific embodiments, the HRSpolypeptide consists or consists essentially of amino acid residues2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO:1.

In some embodiments, at least one endogenous cysteine residue of the HRSpolypeptide has been substituted with another amino acid or deleted. Incertain embodiments, the at least one endogenous cysteine residue isselected from Cys174, Cys191, Cys224, Cys235, Cys507, and Cys509. Inparticular embodiments, the at least one endogenous cysteine residue isselected from Cys224, Cys235, Cys507, and Cys509. In specificembodiments, the endogenous cysteine residues are Cys507 and Cys509. Insome embodiments, all endogenous surface exposed cysteine residues havebeen substituted with another amino acid or deleted.

In certain embodiments, the HRS polypeptide is tandemly repeated. Inparticular embodiments, the HRS polypeptide comprises a WHEP domain. Inspecific embodiments, the HRS polypeptide lacks a functionalaminoacylation domain. In some embodiments, the HRS polypeptide consistsessentially of a WHEP domain. In specific aspects, the WHEP domain of anHRS polypeptide or variant or fragment thereof has the consensussequence in Table D5.

In some embodiments, the Fc region and the HRS polypeptide are separatedby a peptide linker. In certain embodiments, the peptide linker is about1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 aminoacids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 aminoacids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 aminoacids, or 1-5 amino acids in length. In particular embodiments, peptidelinker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,80, 90, or 100 amino acids in length. In certain embodiments, thepeptide linker consists of Gly and/or Ser residues. In some embodiments,the peptide linker is a physiologically stable linker. In otherembodiments, the peptide linker is a releasable linker, optionally anenzymatically-cleavable linker. In specific embodiments, the peptidelinker comprises a sequence of any one of SEQ ID NOS:200-260, or otherpeptide linker described herein.

In some embodiments, the Fc region is fused to the C-terminus of the HRSpolypeptide. In certain embodiments, the Fc region is fused to theN-terminus of the HRS polypeptide.

In certain embodiments, the Fc region comprises one or more of a hinge,CH₂, CH₃, and/or CH₄ domain from a mammalian IgA1, IgA2, IgD, IgE, IgG1,IgG2, IgG3, IgG4, and/or IgM. In some embodiments, the Fc regioncomprises IgG1 hinge, CH₂, and CH₃ domains. In some embodiments, the Fcregion comprises IgG2 hinge, CH₂, and CH₃ domains. In some embodiments,the Fc region comprises IgG3 hinge, CH₂, and CH₃ domains. In particularembodiments, the HRS fusion polypeptide does not comprise the CH₁,C_(L), V_(L), and V_(H) regions of an immunoglobulin.

In specific embodiments, the Fc region comprises any one of SEQ IDNOS:128-163 or 339-342, or a variant, or a fragment, or a combinationthereof. In certain embodiments, the hinge domain is a modified IgG1hinge domain that comprises SEQ ID NO:341.

In particular embodiments, the Fc region comprises an amino acidsequence at least 90% identical to

(SEQ ID NO: 339) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or (SEQ ID NO: 340)SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In certain embodiments, the HRS-Fc fusion polypeptide comprises an aminoacid sequence at least 90% identical to Fc-HRS(2-60) (SEQ ID NO:337), orHRS(1-60)-Fc (SEQ ID NO:338), or Fc-HRS(2-40) (SEQ ID NO:381), orHRS(1-40)-Fc (SEQ ID NO:386), or Fc-HRS(2-45) (SEQ ID NO: 382), orHRS(1-45)-Fc (SEQ ID NO: 387), or Fc-HRS(2-50) (SEQ ID NO: 383), orHRS(1-50)-Fc (SEQ ID NO: 388), or Fc-HRS(2-55) (SEQ ID NO: 384), orHRS(1-55)-Fc (SEQ ID NO: 389), or Fc-HRS(2-66) (SEQ ID NO:385), orHRS(1-66)-Fc (SEQ ID NO:390), or Fc-HRS(2-60) HRS(2-60) (SEQ ID NO:396).

In certain instances, the HRS fusion polypeptide has alteredpharmacokinetics relative to a corresponding HRS polypeptide. Examplesof said altered pharmacokinetics include increased serum half-life,increased bioavailability, increased exposure, and/or decreasedclearance. In certain instances, the exposure is increased by at least100 fold. In some instances, the HRS fusion polypeptide has a half lifeof at least 30 hours in mice. In certain instances, the bioavailabilityis subcutaneous bioavailability that is increased by at least about 30%.In some instances, the HRS fusion polypeptide has altered immuneeffector activity relative to a corresponding HRS polypeptide. Examplesof such immune effector activities include one or more of complementactivation, complement-dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediatedphagocytosis (ADCP).

In certain embodiments, the Fc region comprises a variant Fc region,relative to a wild-type Fc region. In some embodiments, the variant Fcregion comprises a sequence that is at least 90% identical to any one ofSEQ ID NOS:128-163 or 341, or a combination of said sequences. Incertain embodiments, the variant Fc region comprises a hybrid of one ormore Fc regions from different species, different Ig classes, ordifferent Ig subclasses. In particular embodiments, the variant Fcregion comprises a hybrid of one or more hinge, CH₂, CH₃, and/or CH₄domains of Fc regions from different species, different Ig classes,and/or different Ig subclasses.

In certain embodiments, the variant Fc region is a modified glycoform,relative to a corresponding, wild-type Fc region. In particularembodiments, the variant Fc region has altered pharmacokinetics relativeto a corresponding, wild-type Fc region. Examples of such alteredpharmacokinetics include serum half-life, bioavailability, and/orclearance. In some embodiments, the variant Fc region has alteredeffector activity relative to a corresponding, wild-type Fc region.Examples of such effector activities include one or more of complementactivation, complement-dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediatedphagocytosis (ADCP).

In certain embodiments, the variant Fc region has altered binding to oneor more Fcγ receptors, relative to a corresponding, wild-type Fc region.Exemplary Fcγ receptors are described herein and known in the art.

In certain embodiments, the variant Fc region has altered binding to oneor more FcRn receptors, relative to a corresponding, wild-type Fcregion. Exemplary FcRn receptors are described herein and known in theart.

In some embodiments, the variant Fc region has altered (e.g., increased)solubility, relative to a corresponding, wild-type Fc region, and theHRS-Fc fusion polypeptide has altered solubility, relative to acorresponding, unmodified HRS polypeptide.

In specific embodiments, the HRS-Fc fusion polypeptide is substantiallyin dimeric form in a physiological solution, or under otherphysiological conditions, such as in vivo conditions. In specificembodiments, the HRS-Fc fusion polypeptide has substantially the samesecondary structure a corresponding unmodified or differently modifiedHRS polypeptide, as determined via UV circular dichroism analysis.

In some embodiments, the HRS-Fc fusion polypeptide has a plasma or serapharmacokinetic AUC profile at least 5-fold greater than acorresponding, unmodified HRS polypeptide when administered to a mammal.

In certain embodiments, the HRS-Fc fusion polypeptide has substantiallythe same activity of a corresponding unmodified or differently modifiedHRS polypeptide in an assay of anti-inflammatory activity.

In certain embodiments, the HRS-Fc fusion polypeptide has greater than2-fold the activity of a corresponding unmodified or differentlymodified HRS polypeptide in an assay of anti-inflammatory activity.

In certain embodiments, the HRS-Fc fusion polypeptide has a stabilitywhich is at least 30% greater than a corresponding unmodified ordifferently modified HRS polypeptide when compared under similarconditions at room temperature, for 7 days in PBS at pH 7.4.

Specific examples of HRS-Fc fusion polypeptides may comprise at leastone of SEQ ID NO:107-110 or 337-338 or 349-350 or 381-390 or 396, or anamino acid sequence at least 80%, 90%, 95%, 98% identical to SEQ IDNO:107-110 or 337-338 or 349-350 or 381-390 or 396. SEQ ID NOS:107 and338 are the amino acid sequences of exemplary C-terminal Fc fusionpolypeptides to residues 1-60 of SEQ ID NO:1 (HRS(1-60)_Fc); SEQ IDNOS:108 and 337 are the amino acid sequences of exemplary N-terminal Fcfusion polypeptides to residues 1-60 of SEQ ID NO:1 (Fc_HRS(1-60)); SEQID NO:109 is the amino acid sequence of an exemplary C-terminal Fcfusion polypeptide to residues 1-506 of SEQ ID NO:1 (HRS(1-506)_Fc); andSEQ ID NO:110 is the amino acid sequence of an exemplary N-terminal Fcfusion polypeptide to residues 1-506 of SEQ ID NO:1 (Fc_HRS(1-506)).

In some embodiments, the HRS-Fc fusion polypeptide has ananti-inflammatory activity, for example, in a cell-based assay or uponadministration to a subject.

Also included are compositions, for example, pharmaceutical ortherapeutic compositions, comprising a HRS-Fc fusion polypeptidedescribed herein and a pharmaceutically acceptable or pharmaceuticalgrade carrier or excipient. In some compositions, the polypeptide as isat least about 95% pure and less than about 5% aggregated. In someembodiments, the composition is formulated for delivery via oral,subcutaneous, intranasal, pulmonary or parental administration. Incertain embodiments, the composition comprises a delivery vehicleselected from the group consisting of liposomes, micelles, emulsions,and cells.

In some embodiments, the composition is for use in a) treating aninflammatory or autoimmune disease, b) reducing muscle or lunginflammation optionally associated with an autoimmune or inflammatorydisease, c) inducing tolerance to a histidyl-tRNA synthetase (HRS)autoantigen, d) eliminating a set or subset of T cells involved in anautoimmune response to a HRS autoantigen, e) reducing tissueinflammation in a subject, optionally muscle, lung, and/or skin tissue,f) treating a muscular dystrophy, g) treating rhabdomyolysis, musclewasting, cachexia, muscle inflammation, or muscle injury, and/or h)treating a disease associated with an autoantibody.

Also included are dosing regimens which maintain an average steady-stateconcentration of an histidyl-tRNA synthetase (HRS)-Fc fusion polypeptidein a subject's plasma of between about 300 pM and about 1000 nM whenusing a dosing interval of 3 days or longer, comprising administering tothe subject a therapeutic composition or HRS-Fc fusion polypeptidedescribed herein.

Some embodiments include methods for maintaining histidyl-tRNAsynthetase (HRS)-Fc fusion polypeptide levels above the minimumeffective therapeutic level in a subject in need thereof, comprisingadministering to the subject a therapeutic composition or HRS-Fc fusionpolypeptide described herein.

Also included are methods for treating an inflammatory or autoimmunedisease or condition in a subject in need thereof, comprisingadministering to the subject a therapeutic composition or HRS-Fc fusionpolypeptide described herein.

Some embodiments include methods of reducing muscle or lung inflammationassociated with an autoimmune or inflammatory disease in a subject inneed thereof, comprising administering to the subject a therapeuticcomposition or HRS-Fc fusion polypeptide described herein.

Certain embodiments include methods of inducing tolerance to ahistidyl-tRNA synthetase (HRS) autoantigen in a subject in need thereof,comprising administering to the subject a therapeutic composition orHRS-Fc fusion polypeptide described herein.

Some embodiments include methods for eliminating a set or subset of Tcells involved in an autoimmune response to a histidyl-tRNA synthetase(HRS) autoantigen in a subject in need thereof, comprising administeringto the subject a therapeutic composition or HRS-Fc fusion polypeptidedescribed herein.

Also included are methods of reducing tissue inflammation in a subjectin need thereof, comprising administering to the subject a therapeuticcomposition or HRS-Fc fusion polypeptide described herein. In certainembodiments, the tissue is selected from muscle, gut, brain, lung, andskin.

Some embodiments include methods of treating a muscular dystrophy in asubject in need thereof, comprising administering to the subject atherapeutic composition or HRS-Fc fusion polypeptide described herein.In particular embodiments, the muscular dystrophy is selected fromDuchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifussmuscular dystrophy, Limb-girdle muscular dystrophy, facioscapulohumeralmuscular dystrophy, myotonic dystrophy, oculopharyngeal musculardystrophy, distal muscular dystrophy, and congenital muscular dystrophy.

Certain embodiments include methods of treating rhabdomyolysis, musclewasting, cachexia, muscle inflammation, or muscle injury in a subject inneed thereof, comprising administering to the subject a therapeuticcomposition or HRS-Fc fusion polypeptide described herein.

Some embodiments include methods of treating a disease associated withan autoantibody, comprising administering to a subject in need thereof acomposition or AARS/HRS polypeptide described herein. In someembodiments, the disease is selected from the group consisting ofinflammatory myopathies, including inflammatory myopathies,polymyositis, dermatomyositis and related disorders,polymyositis-scleroderma overlap, inclusion body myositis (IBM),anti-synthetase syndrome, interstitial lung disease, arthritis, andReynaud's phenomenon. In some embodiments, the composition isadministered to the subject prior to the appearance of disease symptoms.In some embodiments, the autoantibody is specific for histidyl-tRNAsynthetase. In some embodiments, the HRS polypeptide comprises at leastone epitope of the histidyl-tRNA synthetase recognized by the diseasespecific autoantibody. In some embodiments, the epitope is animmunodominant epitope recognized by antibodies in sera from thesubject. In some embodiments, the HRS polypeptide blocks the binding ofthe autoantibody to native histidyl-tRNA synthetase. In someembodiments, the HRS polypeptide causes clonal deletion of auto-reactiveT-cells. In some embodiments, the HRS polypeptide causes functionalinactivation of the T cells involved in the autoimmune response. In someembodiments, administration of the HRS polypeptide results in reducedmuscle or lung inflammation. In some embodiments, the HRS polypeptideinduces tolerance to an auto-antigen.

In certain embodiments, the composition is formulated for delivery viaoral, intranasal, pulmonary, intramuscular, or parental administration.

Also included are isolated polynucleotides, comprising a nucleotidesequence that encodes a HRS-Fc conjugate or fusion polypeptide describedherein, including vectors that comprise such polynucleotides, and hostcells that comprise said polynucleotides and/or vectors.

Some embodiments include methods for manufacturing a HRS-Fc fusionpolypeptide described herein, comprising a) culturing a host cell (e.g.,E. coli K-12 host cell) to express a HRS-Fc fusion polypeptide, whereinthe host cell comprises a polynucleotide that encodes a HRS-Fc fusionpolypeptide described herein, which is operably linked to a regulatoryelement; and b) isolating the HRS-Fc fusion polypeptide from the hostcell. In specific embodiments, E. coli K-12 strain is selected fromW3110 and UT5600.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biologyand recombinant DNA techniques within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (3^(rd) Edition, 2000);DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); OligonucleotideSynthesis: Methods and Applications (P. Herdewijn, ed., 2004); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic AcidHybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005)Culture of Animal Cells, a Manual of Basic Technique, 5^(th) Ed. HobokenN.J., John Wiley & Sons; B. Perbal, A Practical Guide to MolecularCloning (3^(rd) Edition 2010); Farrell, R., RNA Methodologies: ALaboratory Guide for Isolation and Characterization (3^(rd) Edition2005). Poly(ethylene glycol), Chemistry and Biological Applications,ACS, Washington, 1997; Veronese, F., and J. M. Harris, Eds., Peptide andprotein PEGylation, Advanced Drug Delivery Reviews, 54(4) 453-609(2002); Zalipsky, S., et al., “Use of functionalized Poly(EthyleneGlycols) for modification of polypeptides” in Polyethylene GlycolChemistry: Biotechnical and Biomedical Applications.

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

As used herein, the term “amino acid” is intended to mean both naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics. Naturally occurring amino acids include the 20(L)-amino acids utilized during protein biosynthesis as well as otherssuch as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine, for example. Non-naturallyoccurring amino acids include, for example, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like, which areknown to a person skilled in the art. Amino acid analogs includemodified forms of naturally and non-naturally occurring amino acids.Such modifications can include, for example, substitution or replacementof chemical groups and moieties on the amino acid or by derivatizationof the amino acid. Amino acid mimetics include, for example, organicstructures which exhibit functionally similar properties such as chargeand charge spacing characteristic of the reference amino acid. Forexample, an organic structure which mimics arginine (Arg or R) wouldhave a positive charge moiety located in similar molecular space andhaving the same degree of mobility as the e-amino group of the sidechain of the naturally occurring Arg amino acid. Mimetics also includeconstrained structures so as to maintain optimal spacing and chargeinteractions of the amino acid or of the amino acid functional groups.Those skilled in the art know or can determine what structuresconstitute functionally equivalent amino acid analogs and amino acidmimetics.

As used herein, a subject “at risk” of developing a disease or adversereaction may or may not have detectable disease, or symptoms of disease,and may or may not have displayed detectable disease or symptoms ofdisease prior to the treatment methods described herein. “At risk”denotes that a subject has one or more risk factors, which aremeasurable parameters that correlate with development of a disease, asdescribed herein and known in the art. A subject having one or more ofthese risk factors has a higher probability of developing disease, or anadverse reaction than a subject without one or more of these riskfactor(s).

An “autoimmune disease” as used herein is a disease or disorder arisingfrom and directed against an individual's own tissues. Examples ofautoimmune diseases or disorders include, but are not limited to,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); respiratory distresssyndrome (including adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g., Type I diabetes mellitus or insulin dependent diabetesmellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, inflammatory myopathies,interstitial lung disease, granulomatosis and vasculitis; perniciousanemia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder; multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia, etc.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey materially affect the activity or action of the listed elements.

The term “clonal deletion” refers to the deletion (e.g., loss, or death)of auto-reactive T-cells. Clonal deletion can be achieved centrally inthe thymus, or in the periphery, or both.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule (e.g., HRS polypeptide), to an immunoglobulin Fc region. Oneexample of a conjugate polypeptide is a “fusion protein” or “fusionpolypeptide,” that is, a polypeptide that is created through the joiningof two or more coding sequences, which originally coded for separatepolypeptides; translation of the joined coding sequences results in asingle, fusion polypeptide, typically with functional properties derivedfrom each of the separate polypeptides.

The recitation “endotoxin free” or “substantially endotoxin free”relates generally to compositions, solvents, and/or vessels that containat most trace amounts (e.g., amounts having no clinically adversephysiological effects to a subject) of endotoxin, and preferablyundetectable amounts of endotoxin. Endotoxins are toxins associated withcertain bacteria, typically gram-negative bacteria, although endotoxinsmay be found in gram-positive bacteria, such as Listeria monocytogenes.The most prevalent endotoxins are lipopolysaccharides (LPS) orlipo-oligo-saccharides (LOS) found in the outer membrane of variousGram-negative bacteria, and which represent a central pathogenic featurein the ability of these bacteria to cause disease. Small amounts ofendotoxin in humans may produce fever, a lowering of the blood pressure,and activation of inflammation and coagulation, among other adversephysiological effects.

Therefore, in pharmaceutical production, it is often desirable to removemost or all traces of endotoxin from drug products and/or drugcontainers, because even small amounts may cause adverse effects inhumans. A depyrogenation oven may be used for this purpose, astemperatures in excess of 300° C. are typically required to break downmost endotoxins. For instance, based on primary packaging material suchas syringes or vials, the combination of a glass temperature of 250° C.and a holding time of 30 minutes is often sufficient to achieve a 3 logreduction in endotoxin levels. Other methods of removing endotoxins arecontemplated, including, for example, chromatography and filtrationmethods, as described herein and known in the art. Also included aremethods of producing HRS-Fc conjugates in and isolating them fromeukaryotic cells such as mammalian cells to reduce, if not eliminate,the risk of endotoxins being present in a composition of the invention.Preferred are methods of producing HRS-Fc conjugates in and isolatingthem from serum free cells.

Endotoxins can be detected using routine techniques known in the art.For example, the Limulus Amoebocyte Lysate assay, which utilizes bloodfrom the horseshoe crab, is a very sensitive assay for detectingpresence of endotoxin. In this test, very low levels of LPS can causedetectable coagulation of the limulus lysate due a powerful enzymaticcascade that amplifies this reaction. Endotoxins can also be quantitatedby enzyme-linked immunosorbent assay (ELISA). To be substantiallyendotoxin free, endotoxin levels may be less than about 0.001, 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/ml. Typically, 1 nglipopolysaccharide (LPS) corresponds to about 1-10 EU.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., Nucleic Acids Research. 12, 387-395, 1984), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

A “physiologically stable” linker refers to a linker that issubstantially stable in water or under physiological conditions (e.g.,in vivo, in vitro culture conditions, for example, in the presence ofone or more proteases), that is to say, it does not undergo adegradation reaction (e.g., enzymatically degradable reaction) underphysiological conditions to any appreciable extent over an extendedperiod of time. Generally, a physiologically stable linker is one thatexhibits a rate of degradation of less than about 0.5%, about 1%, about2%, about 3%, about 4%, or about 5% per day under physiologicalconditions.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated peptide” or an “isolated polypeptide” and thelike, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

The term “half maximal effective concentration” or “EC₅₀” refers to theconcentration of a HRS-Fc conjugate described herein at which it inducesa response halfway between the baseline and maximum after some specifiedexposure time; the EC₅₀ of a graded dose response curve thereforerepresents the concentration of a compound at which 50% of its maximaleffect is observed. In certain embodiments, the EC₅₀ of an agentprovided herein is indicated in relation to a “non-canonical” activity,as noted above. EC₅₀ also represents the plasma concentration requiredfor obtaining 50% of a maximum effect in vivo. Similarly, the “EC₉₀”refers to the concentration of an agent or composition at which 90% ofits maximal effect is observed. The “EC₉₀” can be calculated from the“EC₅₀” and the Hill slope, or it can be determined from the datadirectly, using routine knowledge in the art. In some embodiments, theEC₅₀ of a HRS-Fc conjugate is less than about 0.01, 0.05, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nM.Preferably, biotherapeutic composition will have an EC₅₀ value of about1 nM or less.

The “half-life” of a HRS-Fc conjugate can refer to the time it takes forthe conjugate to lose half of its pharmacologic, physiologic, or otheractivity, relative to such activity at the time of administration intothe serum or tissue of an organism, or relative to any other definedtime-point. “Half-life” can also refer to the time it takes for theamount or concentration of a HRS-Fc conjugate to be reduced by half of astarting amount administered into the serum or tissue of an organism,relative to such amount or concentration at the time of administrationinto the serum or tissue of an organism, or relative to any otherdefined time-point. The half-life can be measured in serum and/or anyone or more selected tissues.

The term “linkage,” “linker,” “linker moiety,” or “L” is used herein torefer to a linker that can be used to separate a HRS polypeptides fromanother HRS polypeptide and/or from one or more Fc regions. The linkermay be physiologically stable or may include a releasable linker such asan enzymatically degradable linker (e.g., proteolytically cleavablelinkers). In certain aspects, the linker may be a peptide linker, forinstance, as part of a HRS-Fc fusion protein. In some aspects, thelinker may be a non-peptide linker.

The terms “modulating” and “altering” include “increasing,” “enhancing”or “stimulating,” as well as “decreasing” or “reducing,” typically in astatistically significant or a physiologically significant amount ordegree relative to a control. An “increased,” “stimulated” or “enhanced”amount is typically a “statistically significant” amount, and mayinclude an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30 or more times (e.g., 500, 1000 times) (including all integers anddecimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.)the amount produced by no composition (e.g., in the absence of any ofthe HRS-Fc conjugates of the invention) or a control composition, sampleor test subject. A “decreased” or “reduced” amount is typically a“statistically significant” amount, and may include a 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% decrease in the amount produced by no composition (theabsence of an agent or compound) or a control composition, including allintegers in between. As one non-limiting example, a control in comparingcanonical and non-canonical activities could include the HRS-Fcconjugate of interest compared to a corresponding (sequence-wise),unmodified or differently modified HRS polypeptide. Other examples ofcomparisons and “statistically significant” amounts are describedherein.

“Non-canonical” activity as used herein, refers generally to either i) anew, non-aminoacylation activity possessed by HRS polypeptide of theinvention that is not possessed to any significant degree by the intactnative full length parental protein, or ii) an activity that waspossessed by the by the intact native full length parental protein,where the HRS polypeptide either exhibits a significantly higher (e.g.,at least 20% greater) specific activity with respect to thenon-canonical activity compared to the intact native full lengthparental protein, or exhibits the activity in a new context; for exampleby isolating the activity from other activities possessed by the intactnative full length parental protein. In the case of HRS polypeptides,non-limiting examples of non-canonical activities include extracellularsignaling including the modulation of cell proliferation, modulation ofcell migration, modulation of cell differentiation (e.g., hematopoiesis,neurogenesis, myogenesis, osteogenesis, and adipogenesis), modulation ofgene transcription, modulation of apoptosis or other forms of celldeath, modulation of cell signaling, modulation of cellular uptake, orsecretion, modulation of angiogenesis, modulation of cell binding,modulation of cellular metabolism, modulation of cytokine production oractivity, modulation of cytokine receptor activity, modulation ofinflammation, immunogenicity, and the like.

In certain embodiments, the “purity” of any given agent (e.g., HRS-Fcconjugate such as a fusion protein) in a composition may be specificallydefined. For instance, certain compositions may comprise an agent thatis at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%pure, including all decimals in between, as measured, for example and byno means limiting, by high pressure liquid chromatography (HPLC), awell-known form of column chromatography used frequently in biochemistryand analytical chemistry to separate, identify, and quantify compounds.

Without wishing to be bound to any particular theory, an “enzymaticallydegradable linker” means a linker, e.g., amino acid sequence that issubject to degradation by one or more enzymes, e.g., peptidases orproteases.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues and to variants and syntheticanalogues of the same. Thus, these terms apply to amino acid polymers inwhich one or more amino acid residues are synthetic non-naturallyoccurring amino acids, such as a chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally-occurring aminoacid polymers.

A “releasable linker” includes, but is not limited to, a physiologicallycleavable linker and an enzymatically degradable linker. Thus, a“releasable linker” is a linker that may undergo either spontaneoushydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed,acid-catalyzed, base-catalyzed, and so forth) under physiologicalconditions. For example, a “releasable linker” can involve anelimination reaction that has a base abstraction of a proton, (e.g., anionizable hydrogen atom, Ha), as the driving force. For purposes herein,a “releasable linker” is synonymous with a “degradable linker.” Inparticular embodiments, a releasable linker has a half life at pH 7.4,25° C., e.g., a physiological pH, human body temperature (e.g., invivo), of about 30 minutes, about 1 hour, about 2 hour, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours,or about 96 hours or more.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

The term “solubility” refers to the property of a HRS-Fc conjugatepolypeptide provided herein to dissolve in a liquid solvent and form ahomogeneous solution. Solubility is typically expressed as aconcentration, either by mass of solute per unit volume of solvent (g ofsolute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity,molality, mole fraction or other similar descriptions of concentration.The maximum equilibrium amount of solute that can dissolve per amount ofsolvent is the solubility of that solute in that solvent under thespecified conditions, including temperature, pressure, pH, and thenature of the solvent. In certain embodiments, solubility is measured atphysiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0,or pH 7.4. In certain embodiments, solubility is measured in water or aphysiological buffer such as PBS or NaCl (with or without NaP). Inspecific embodiments, solubility is measured at relatively lower pH(e.g., pH 6.0) and relatively higher salt (e.g., 500 mM NaCl and 10 mMNaP). In certain embodiments, solubility is measured in a biologicalfluid (solvent) such as blood or serum. In certain embodiments, thetemperature can be about room temperature (e.g., about 20, 21, 22, 23,24, 25° C.) or about body temperature (37° C.). In certain embodiments,a HRS-Fc conjugate polypeptide has a solubility of at least about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at roomtemperature or at 37° C.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treated ordiagnosed with a HRS-Fc conjugate polypeptide of the invention. Suitablesubjects (patients) include laboratory animals (such as mouse, rat,rabbit, or guinea pig), farm animals, and domestic animals or pets (suchas a cat or dog). Non-human primates and, preferably, human patients,are included.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition, and may includeeven minimal changes or improvements in one or more measurable markersof the disease or condition being treated. “Treatment” or “treating”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof. The subjectreceiving this treatment is any subject in need thereof. Exemplarymarkers of clinical improvement will be apparent to persons skilled inthe art.

Histidyl-tRNA Synthetase Derived Polypeptides

Embodiments of the present invention relate to histidyl-tRNA synthetasepolypeptide (“HRS or HisRS polypeptides”)-Fc conjugates, includingHRS-Fc conjugates that comprise wild-type HRS sequences,naturally-occurring sequences, non-naturally occurring sequences, and/orvariants and fragments thereof. Specific examples of HRS derivedpolypeptides include those with altered cysteine content. Histidyl-tRNAsynthetases belong to the class II tRNA synthetase family, which hasthree highly conserved sequence motifs. Class I and II tRNA synthetasesare widely recognized as being responsible for the specific attachmentof an amino acid to its cognate tRNA in a two-step reaction: the aminoacid (AA) is first activated by ATP to form AA-AMP and then transferredto the acceptor end of the tRNA. The full length histidyl-tRNAsynthetases typically exist either as a cytosolic homodimer, or analternatively spliced mitochondrial form.

More recently it has been established that some biological fragments, oralternatively spliced isoforms of eukaryotic histidyl-tRNA synthetases(Physiocrines, or HRS polypeptides), or in some contexts the intactsynthetase, modulate certain cell-signaling pathways, or haveanti-inflammatory properties. These activities, which are distinct fromthe classical role of tRNA synthetases in protein synthesis, arecollectively referred to herein as “non-canonical activities.” ThesePhysiocrines may be produced naturally by either alternative splicing orproteolysis, and can act in a cell autonomous fashion (i.e., within thehost cell) or a non-cell autonomous fashion (i.e., outside the hostcell) to regulate a variety of homeostatic mechanisms. For example, asprovided in the present invention, HRS polypeptides such as theN-terminal fragment of histidyl-tRNA synthetase (e.g., HRS 1-48, HRS1-60) are capable, inter alia, of exerting an anti-inflammatory signalby blocking the migration, activation, or differentiation ofinflammatory cells (e.g., monocytes, macrophages, T cells, B cells)associated with the sites of active inflammation in vivo. In addition,certain mutations or deletions (e.g., HRS 1-506, HRS 1-60) relative tothe full-length HRS polypeptide sequence confer increased activitiesand/or improved pharmacological properties. The sequences of certainexemplary HRS polypeptides are provided in Table D1.

TABLE D1 Exemplary HRS polypeptides Type/ species/ SEQ ID Name ResiduesAmino acid and Nucleic Acid Sequences NO: N-terminal PhysiocrinesFL cytosolic Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  1wild type Human/ LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQPLCIC FL Protein/MPLLGLLPRRAWASLLSQLLRPPCASCTGAVRCQSQVAEAV  2 mitochondrial Human/LTSQLKAHQEKPNFIIKTPKGTRDLSPQHMVVREKILDLVISC wild typeFKRHGAKGMDTPAFELKETLTEKYGEDSGLMYDLKDQGGELLSLRYDLTVPFARYLAMNKVKKMKRYHVGKVWRRESPTIVQGRYREFCQCDFDIAGQFDPMIPDAECLKIMCEILSGLQLGDFLIKVNDRRIVDGMFAVCGVPESKFRAICSSIDKLDKMAWKDVRHEMVVKKGLAPEVADRIGDYVQCHGGVSLVEQMFQDPRLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPTQAGEEPLNVGSVAAGGRYDGLVGMFDPKGHKVPCVGLSIGVERIFYIVEQRMKTKGEKVRTTETQVFVATPQKNFLQERLKLIAELWDSGIKAEMLYKNNPKLLTQLHYCESTGIPLVVIIGEQELKEGVIKIRSVASREEVAIKREN FVAEIQKRLSES HisRS1^(N1)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  3 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-141RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGG ELLSLRYDLTVPFARYLAMHisRS1^(N2) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  4 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-408RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTE HisRS1^(N3) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  5 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-113RCFKRHGAEVIDTPVFELKETLMGKYGEDSKL HisRS1^(N4) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  6 Human/ LKAQLGPDESKQKFVLKTPK1-60 HisRS1^(N5) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  7Human/ LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-243 +RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGG 27 aaELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVGYP WWNSCSRILNYPKTSRPWRAWETC-terminal Physiocrines Type/ species/ SEQ ID Name ResiduesAmino acid and Nucleic Acid Sequences NO. HisRS1^(C1) Protein/RTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKK  8 Human/NPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREE 405-509VDVRREDLVEEIKRRTGQPLCIC HisRS1^(C2) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  9 Human/LKAQLGPDESKQKFVLKTPKDFDIAGNFDPMIPDAECLKIM 1-60 + 175-CEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSV 509DKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSV TSREEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C3) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 10 Human/LKAQLGPDESKQKFVLKTPKVNDRRILDGMFAICGVSDSK 1-60 + 211-FRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDY 509VQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCIC HisRS1^(C4) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 11 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 1-100 +IRCFKRHGAEVIDTPVFELKVNDRRILDGMFAICGVSDSKF 211-509RTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCIC HisRS1^(C5) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 12 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 1-174 +IRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQG 211-509GELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSV TSREEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C6) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 13 Human/LKAQLGPDESKQKFVLKTPKETLMGKYGEDSKLIYDLKDQ 1-60 + 101-GGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDN 509PAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTIETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREE VDVRREDLVEEIKRRTGQPLCICHisRS1^(C7) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 14 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 1-100 +IRCFKRHGAEVIDTPVFELKDFDIAGNFDPMIPDAECLKIMC 175-509EILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSV TSREEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C8) Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 15 Human/LKAQLGPDESKQKFVLKTPKALEEKIRTIETQVLVASAQK 1-60 + 399-KLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEA 509GIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKR RTGQPLCIC HisRS1^(C9)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 16 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 1-100 +IRCFKRHGAEVIDTPVFELKALEEKIRTIETQVLVASAQKK 399-509LLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRR TGQPLCIC HisRS1^(C10)Protein/ MFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQ 17 Human/VLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLN 369-509QLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRRE DLVEEIKRRTGQPLCICInternal Physiocrines Type/ ID species/ SEQ  Name ResiduesAmino acid and Nucleic Acid Sequences NO. HisRS1^(I1) Protein/CLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFR 18 Human/TICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYV 191-333QQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTL FGIDDKISFDLSLARGLDYYTG

A number of naturally occurring histidyl-tRNA synthetase singlenucleotide polymorphisms (SNPs) and naturally occurring variants of thehuman gene have been sequenced, and are known in the art to be at leastpartially functionally interchangeable. Several such variants ofhistidyl-tRNA synthetase (i.e., representative histidyl-tRNA synthetaseSNPs) are shown in Table D2.

TABLE D2 Human Histidyl tRNA synthetase SNPs Gene Bank Nucleo- Gene BankAccession tide Accession Nucleotide Number Change Number Changers193103291 A/G rs186312047 A/G rs192923161 C/T rs186176857 C/Trs192784934 A/G rs186043734 C/G rs192164884 A/G rs185867584 C/Trs192090865 A/C rs185828130 A/G rs192015101 A/T rs185537686 A/Grs191999492 A/G rs185440931 C/T rs191852363 C/T rs185100584 A/Crs191532032 A/T rs185077558 C/T rs191391414 C/T rs184748736 C/Grs191385862 A/G rs184591417 C/T rs191205977 A/G rs184400035 C/Grs191104160 A/G rs184098206 C/T rs190989313 C/G rs183982931 C/Trs190818970 A/T rs183942045 A/G rs190476138 C/T rs183854085 A/Grs190289555 C/T rs183430882 G/T rs190065567 A/G rs183419967 A/Crs189624055 C/T rs183366286 A/G rs189563577 G/T rs183084050 C/Trs189404434 A/G rs182948878 C/T rs189268935 A/G rs182813126 A/Grs189103453 A/T rs182498374 A/G rs188839103 A/G rs182161259 A/Trs188766717 A/G rs182119902 C/T rs188705391 A/G rs182106891 C/Trs188490030 A/G rs181930530 A/G rs188345926 C/T rs181819577 A/Grs188174426 A/G rs181706697 C/T rs187897435 C/T rs181400061 G/Trs187880261 A/G rs181240610 G/T rs187729939 G/T rs181150977 A/Crs187617985 A/T rs180848617 A/G rs187344319 C/T rs180765564 A/Grs187136933 C/T rs151330569 C/G rs186823043 C/G rs151258227 C/Trs186764765 C/T rs151174822 C/T rs186663247 A/G rs150874684 C/Trs186526524 A/G rs150589670 A/G rs150274370 C/T rs145059663 C/Trs150090766 A/G rs144588417 C/T rs149977222 A/G rs144457474 A/Grs149821411 C/T rs144322728 C/T rs149542384 A/G rs143897456 -/Crs149336018 C/G rs143569397 G/T rs149283940 C/T rs143476664 C/Trs149259830 C/T rs143473232 C/G rs149241235 C/T rs143436373 G/Trs149018062 C/T rs143166254 A/G rs148935291 C/T rs143011702 C/Grs148921342 -/A rs142994969 A/G rs148614030 C/T rs142880704 A/Grs148584540 C/T rs142630342 A/G rs148532075 A/C rs142522782 -/AAACrs148516171 C/T rs142443502 C/T rs148394305 -/AA rs142305093 C/Trs148267541 C/T rs142289599 A/G rs148213958 C/T rs142088963 A/Crs147637634 A/G rs141765732 A/C rs147372931 A/C/G rs141386881 A/Trs147350096 A/C rs141291994 A/G rs147288996 C/T rs141285041 C/Trs147194882 G/T rs141220649 C/T rs147185134 C/T rs141147961 -/Crs147172925 A/G rs141123446 -/A rs147011612 C/T rs140516034 A/Grs147001782 A/G rs140169815 C/T rs146922029 C/T rs140005970 G/Trs146835587 A/G rs139699964 C/T rs146820726 C/T rs139555499 A/Grs146801682 C/T rs139447495 C/T rs146571500 G/T rs139364834 -/Ars146560255 C/T rs139362540 A/G rs146205151 -/A rs139300653 -/Ars146159952 A/G rs139251223 A/G rs145532449 C/G rs139145072 A/Grs145446993 A/G rs138612783 A/G rs145112012 G/T rs138582560 A/Grs138414368 A/G rs111863295 C/T rs138377835 A/G rs111519226 C/Grs138300828 C/T rs111314092 C/T rs138067637 C/T rs80074170 A/Trs138035024 A/G rs79408883 A/C rs137973748 C/G rs78741041 G/Trs137917558 A/G rs78677246 A/T rs117912126 A/T rs78299006 A/Grs117579809 G/T rs78085183 A/T rs116730458 C/T rs77844754 C/Trs116411189 A/C rs77585983 A/T rs116339664 C/T rs77576083 A/Grs116203404 A/T rs77154058 G/T rs115091892 G/T rs76999025 A/Grs114970855 A/G rs76496151 C/T rs114176478 A/G rs76471225 G/Trs113992989 C/T rs76085408 G/T rs113720830 C/T rs75409415 A/Grs113713558 A/C rs75397255 C/G rs113627177 G/T rs74336073 A/Grs113489608 A/C rs73791750 C/T rs113408729 G/T rs73791749 A/Trs113255561 A/G rs73791748 C/T rs113249111 C/T rs73791747 A/Trs113209109 A/G rs73273304 C/T rs113066628 G/T rs73271596 C/Trs112967222 C/T rs73271594 C/T rs112957918 A/T rs73271591 A/Grs112859141 A/G rs73271586 A/T rs112769834 C/G rs73271585 A/Grs112769758 A/C rs73271584 A/G rs112701444 A/C rs73271581 C/Trs112585944 A/G rs73271578 A/T rs112439761 A/G rs72800925 G/Trs112427345 A/C rs72800924 C/T rs112265354 C/T rs72800922 A/Trs112113896 C/G rs72432753 -/A rs112033118 C/T rs72427948 -/Ars112029988 A/G rs72388191 -/A rs72317985 -/A rs6873628 C/T rs71583608G/T rs5871749 -/C rs67251579 -/A rs4334930 A/T rs67180750 -/A rs3887397A/G rs63429961 A/T rs3776130 A/C rs61093427 C/T rs3776129 C/T rs61059042-/A rs3776128 A/G rs60936249 -/AA rs3177856 A/C rs60916571 -/A rs2563307A/G rs59925457 C/T rs2563306 A/G rs59702263 -/A rs2563305 C/T rs58302597C/T rs2563304 A/G rs57408905 A/T rs2530242 C/G rs35790592 A/C rs2530241A/G rs35609344 -/A rs2530240 A/G rs35559471 -/A rs2530239 A/G rs35217222-/C rs2530235 A/C rs34903998 -/A rs2230361 C/T rs34790864 C/G rs2073512C/T rs34732372 C/T rs1131046 C/T rs34291233 -/C rs1131045 C/G rs34246519-/T rs1131044 C/T rs34176495 -/C rs1131043 C/G rs13359823 A/G rs1131042A/C rs13180544 A/C rs1131041 C/G rs12653992 A/C rs1131040 A/G rs12652092A/G rs1131039 C/T rs11954514 A/C rs1131038 A/G rs11745372 C/T rs1131037A/G rs11548125 A/G rs1131036 A/G rs11548124 C/G rs1131035 C/T rs11344157-/C rs1131034 A/G rs11336085 -/A rs1131033 A/G rs11318345 -/A rs1131032A/G rs11309606 -/A rs1089305 A/G rs10713463 -/A rs1089304 A/C rs7706544C/T rs1065342 A/C rs7701545 A/T rs1050252 C/T rs6880190 C/T rs1050251A/T rs1050250 A/G rs145769024 -/AAACAAAACAAAACA (SEQ ID NO: 164)rs1050249 C/T rs10534452 -/AAAAC rs1050248 A/C/T rs10534451 -/AAACAAAACA(SEQ ID NO: 165) rs1050247 C/T rs59554063 -/CAAAACAAAA (SEQ ID NO: 166)rs1050246 C/G rs58606188 -/CAAAACAAAACAAAA (SEQ ID NO:167) rs1050245 C/Trs71835204 (LARGEDELETION)/- rs1050222 C/T rs71766955 (LARGEDELETION)/-rs813897 A/G rs144998196 -/AAACAAAACA (SEQ ID NO: 168) rs812381 C/Grs68038188 -/ACAAAACAAA (SEQ ID NO: 169) rs811382 C/T rs71980275 -/AAAACrs801189 C/T rs71848069 -/AAAC rs801188 A/C rs60987104 -/AAAC rs801187A/T rs801185 C/T rs801186 A/G rs702396 C/G

Additionally homologs and orthologs of the human gene exist in otherspecies, as listed in Table D3, and it would thus be a routine matter toselect a naturally occurring amino acid, or nucleotide variant presentin a SNP, or other naturally occurring homolog in place of any of thehuman HRS polypeptide sequences listed in Tables D1, D4-D6, or D8.

TABLE D3 Homologs of Human Histidyl tRNA synthetase Type/species/ SEQ IDResidues Amino acid Sequences NO: Mus musculusMADRAALEELVRLQGAHVRGLKEQKASAEQIEEEVTKLLKLKAQL 19GQDEGKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQFDPMIPDAECLKIMCEILSSLQIGNFLVKVNDRRILDGMFAVCGVPDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQAVEGLGDLKLLFEYLILFGIDDKISFDLSLARGLDYYTGVIYEAVLLQMPTQAGEEPLGVGSIAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEASEEKVRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYWEEAGIPLVAIIGEQELRDGVIKLRSVASREEVDVRREDLVEEIRRRTNQPLSTC Canis lupusMAERAALEELVRLQGERVRGLKQQKASAEQIEEEVAKLLKLKAQLG 20 familiarisPDEGKQKFVLKTPKGTRDYSPRQMAVREKVFDVIISCFKRHGAEVIDTPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQFDPMIPDAECLEIMCEILRSLQIGDFLVKVNDRRILDGMFAICGVPDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADHIGDYVQQHGGISLVEQLLQDPELSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPVQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEATEEKVRTTETQVLVASAQKKLLEERLKLVSELWNAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVASREEVDVPREDLVEEIKRRTSQPFCIC Bos taurusMADRAALEDLVRVQGERVRGLKQQKASAEQIEEEVAKLLKLKAQL 21GPDEGKPKFVLKTPKGTRDYSPRQMAVREKVFDVIISCFKRHGAEVIDTPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQFDPMLPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVPDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQPPARAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKVRTTETQVLVASAQKKLLEERLKLISELWDAGIKAELLYKKNPKLLNQLQYCEETGIPLVAIIGEQELKDGVIKLRSVASREEVDVRREDLVEEIKRRTSQPLCIC RattusMADRAALEELVRLQGAHVRGLKEQKASAEQIEEEVTKLLKLKAQL 22 norvegicusGHDEGKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQFDPMIPDAECLKIMCEILSSLQIGNFQVKVNDRRILDGMFAVCGVPDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQAVEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQMPTQAGEEPLGVGSIAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQKLEASEEKVRTTETQVLVASAQKKLLEERLKLISELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIRRRTSQPLSM Gallus gallusMADEAAVRQQAEVVRRLKQDKAEPDEIAKEVAKLLEMKAHLGGD 23EGKHKFVLKTPKGTRDYGPKQMAIRERVFSAIIACFKRHGAEVIDTPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKITNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQFDPMIPDAECLKIVQEILSDLQLGDFLIKVNDRRILDGMFAVCGVPDSKFRTICSSVDKLDKMPWEEVRNEMVGEKGLSPEAADRIGEYVQLHGGMDLIEQLLQDPKLSQNKLVKEGLGDMKLLFEYLTLFGITGKISFDLSLARGLDYYTGVIYEAVLLQQNDHGEESVSVGSVAGGGRYDGLVGMFDPKGRKVPCVGISIGIERIFSILEQRVEASEEKIRTTETQVLVASAQKKLLEERLKLISELWDAGIKAEVLYKKNPKLLNQLQYCEDTGIPLVAIVGEQELKDGVVKLRVVATGEEVNIRRESLVEEIRRRTNQL Danio rerioMAALGLVSMRLCAGLMGRRSAVRLHSLRVCSGMTISQIDEEVARLL 24QLKAQLGGDEGKHVFVLKTAKGTRDYNPKQMAIREKVFNIIINCFKRHGAETIDSPVFELKETLTGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLANINKITNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGQYDAMIPDAECLKLVYEILSELDLGDFRIKVNDRRILDGMFAICGVPDEKFRTICSTVDKLDKLAWEEVKKEMVNEKGLSEEVADRIRDYVSMQGGKDLAERLLQDPKLSQSKQACAGITDMKLLFSYLELFQITDKVVFDLSLARGLDYYTGVIYEAILTQANPAPASTPAEQNGAEDAGVSVGSVAGGGRYDGLVGMFDPKAGKCPVWGSALALRGSSPSWSRRQSC LQRRCAPLKLKCLWLQHRRTF

Accordingly, in any of the methods therapeutic compositions and kits ofthe invention, the terms “HRS polypeptide” “HRS protein” or “HRS proteinfragment” includes all naturally-occurring and synthetic forms of thehistidyl-tRNA synthetase that possesses a non canonical activity, suchas an anti-inflammatory activity and/or retains at least one epitopewhich specifically cross reacts with an auto-antibody or auto reactiveT-cell from a subject with a disease associated with autoantibodies tohistidyl-tRNA synthetase. Such HRS polypeptides include the full lengthhuman protein, as well as the HRS peptides derived from the full lengthprotein listed in Tables D1, D3-D6, or D8. In some embodiments, the termHRS polypeptide refers to a polypeptide sequence derived from humanhistidyl-tRNA synthetase (SEQ ID NO:1 in Table D1) of about 45 or 50 toabout 250 amino acids in length. It will be appreciated that in any ofHRS-Fc conjugates described herein the N-terminal acid of the HRSpolypeptide (for example, the N-terminal Met) may be deleted from any ofthe sequences listed in Tables D1, D3-D6, or D8 when creating the fusionprotein or conjugate.

In some embodiments, the HRS polypeptide is between about 20-509,20-508, 20-507, 50-506, 20-505, 50-504, 20-503, 20-502, 20-501, 20-500,20-400, 20-300, 20-250, 20-200, or 20-100 amino acids in length. Forinstance, in specific embodiments the polypeptide is between about20-25, 20-35, 20-40, 20-45, 20-55, 20-60, 20-65, 20-70, 20-75, 20-80,20-85, 20-90, 20-95, or 20-100 amino acids in length, or about 30-35,30-40, 30-45, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90,30-95, or 30-100 amino acids in length, or about 40-45, 40-55, 40-60,40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, or 40-100 amino acidsin length, or about 45-50, 45-55, 50-55, 50-60, 50-65, 50-70, 50-75,50-80, 50-85, 50-90, 50-95, or 50-100 amino acids in length, or about60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, or 60-100 amino acidsin length, or about 70-75, 70-80, 70-85, 70-90, 70-95, or 70-100 aminoacids in length, or about 80-85, 80-90, 80-95, or 80-100 amino acids inlength. In certain embodiments, the HRS polypeptide is about 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 501,502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length.

In some embodiments, the HRS polypeptide does not significantly competefor disease associated auto-antibody binding (e.g., Jo-1 antibody) towild type histidyl-tRNA synthetase in a competitive ELISA up to aconcentration of about 1 to 5×10⁻⁷M, or higher. Accordingly, in someembodiments, the HRS polypeptide has a lower affinity to diseaseassociated auto-antibody than wild type histidyl-tRNA synthetase (SEQ IDNO:1) as measured in a competitive ELISA. In some embodiments, the HRSpolypeptide has an apparent affinity for the disease associatedauto-antibody (e.g., Jo-1 antibody) which is at least about 10 foldless, or at least about 20 fold less, or at least about 50 fold less, orat least about 100 fold less than the affinity of the disease associatedauto-antibody to wild type human (SEQ ID NO:1).

Thus all such homologues, orthologs, and naturally-occurring, orsynthetic isoforms of histidyl-tRNA synthetase (e.g., any of theproteins listed in Tables D1, D3-D6, or D8) are included in any of themethods, HRS-Fc conjugates, kits and compositions of the invention, aslong as they retain at least one epitope which specifically cross reactswith an auto-antibody or auto reactive T-cell from a subject with adisease associated with autoantibodies to histidyl tRNA synthetase, orpossess a non canonical activity. The HRS polypeptides may be in theirnative form, i.e., as different variants as they appear in nature indifferent species which may be viewed as functionally equivalentvariants of human histidyl -tRNA synthetase, or they may be functionallyequivalent natural derivatives thereof, which may differ in their aminoacid sequence, e.g., by truncation (e.g., from the N- or C-terminus orboth) or other amino acid deletions, additions, insertions,substitutions, or post-translational modifications. Naturally-occurringchemical derivatives, including post-translational modifications anddegradation products of any HRS polypeptide, are also specificallyincluded in any of the methods and compositions of the inventionincluding, e.g., pyroglutamyl, iso-aspartyl, proteolytic,phosphorylated, glycosylated, oxidatized, isomerized, and deaminatedvariants of a HRS polypeptide or HRS-Fc conjugate. HRS polypeptides andHRS-Fc conjugates can also be composed of naturally-occurring aminoacids and/or non-naturally occurring amino acids, as described herein.

As noted above, embodiments of the present invention include allhomologues, orthologs, and naturally-occurring isoforms of histidyl-tRNAsynthetase (e.g., any of the proteins listed in or derivable from, ortheir corresponding nucleic acids listed in, the Tables or the SequenceListing) and “variants” of these HRS reference polypeptides. Therecitation polypeptide “variant” refers to polypeptides that aredistinguished from a reference HRS polypeptide by the addition,deletion, and/or substitution of at least one amino acid residue, andwhich typically retain (e.g., mimic) or modulate (e.g., antagonize) oneor more non-canonical activities of a reference HRS polypeptide.Variants also include polypeptides that have been modified by theaddition, deletion, and/or substitution of at least one amino acidresidue to have improved stability or other pharmaceutical properties.

In certain embodiments, a polypeptide variant is distinguished from areference polypeptide by one or more substitutions, which may beconservative or non-conservative, as described herein and known in theart. In certain embodiments, the polypeptide variant comprisesconservative substitutions and, in this regard, it is well understood inthe art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide. In some embodiments, the variant comprises one or moreconserved residues, including one or more of Leu7, Gln14, Gly15, Val18,Arg19, Leu21, Lys22, Lys25, Ala26, Val35, Leu38, Leu39, Leu41, and Lys42 (based on the numbering of SEQ ID NO:1).

Specific examples of HRS polypeptide variants useful in any of themethods and compositions of the invention include full-length HRSpolypeptides, or truncations or splice variants thereof (e.g., any ofthe proteins listed in or derivable from the Tables or Sequence Listing)which i) retain detectable non canonical activity and/or retain at leastone epitope which specifically cross reacts with an auto-antibody orauto reactive T-cell from a subject with a disease associated withautoantibodies to histidyl-tRNA synthetase, and ii) have one or moreadditional amino acid insertions, substitutions, deletions, and/ortruncations. In certain embodiments, a variant polypeptide includes anamino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequenceidentity or similarity to a corresponding sequence of a HRS referencepolypeptide, as described herein, (e.g., any of the proteins listed inor derivable from the Tables or Sequence Listing) and substantiallyretains the non-canonical activity of that reference polypeptide. Alsoincluded are sequences differing from the reference HRS sequences by theaddition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150 or more amino acids but which retain theproperties of the reference HRS polypeptide. In certain embodiments, theamino acid additions or deletions occur at the C-terminal end and/or theN-terminal end of the HRS reference polypeptide. In certain embodiments,the amino acid additions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more wild-type residues(i.e., from the corresponding full-length HRS polypeptide) that areproximal to the C-terminal end and/or the N-terminal end of the HRSreference polypeptide.

In some embodiments, the HRS polypeptides comprise a polypeptidefragment of the full length histidyl tRNA synthetase of about 45 to 250or about 50 to 250 amino acids, which comprises, consists, or consistsessentially of the amino acids of the HRS polypeptide sequence set forthin one or more of SEQ ID NOS:1-106, 170-181, or 185-191. In someembodiments, the HRS polypeptide comprises, consists, or consistsessentially of residues 1-141, 1-408, 1-113, or 1-60 of SEQ ID NO:1. Insome aspects, the HRS polypeptide is a splice variant that comprises,consists, or consists essentially of residues 1-60+175-509, 1-60+211-509or 1-60+101-509 of SEQ ID NO:1. In particular aspects, the HRSpolypeptide comprises, consists, or consists essentially of residues1-48 or 1-506 of SEQ ID NO:1.

In certain embodiments, a HRS polypeptide of the invention comprises,consists, or consists essentially of the minimal active fragment of afull-length HRS polypeptide capable of modulating an anti-inflammatoryactivity in vivo or having antibody or auto-reactive T-cell blockingactivities. In one aspect, such a minimal active fragment comprises,consists, or consists essentially of the WHEP domain, (i.e., about aminoacids 1-43 of SEQ ID NO:1). In some aspects, the minimal active fragmentcomprises, consists, or consists essentially of the aminoacylationdomain, (i.e., about amino acids 54-398 of SEQ ID NO:1). In someaspects, the minimal active fragment comprises, consists, or consistsessentially of the anticodon binding domain (i.e., about amino acids406-501 of SEQ ID NO:1). Other exemplary active fragments are shown inTable D4 below.

TABLE D4 Exemplary HRS polypeptide fragments Amino Acid Residue Range ofSEQ SEQ ID ID Name NO: 1 Amino acid sequence NO: HRS(1-500) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 170 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-500RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKR HRS(1-501) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 171 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-501RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRR HRS(1-502) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 172 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-502RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRT HRS(1-503) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 173 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-503RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTG HRS(1-504) Protein/MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 174 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-504RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQ HRS(1-505)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 175 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-505RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQP HRS1^(N8)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  25 HRS(1-506) Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-506RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQPL HRS(1-507)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 176 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-507RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQPLC HRS(1-508)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 177 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-508RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQPLCI HRS(1-509)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 178 Human/LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII 1-509RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDL VEEIKRRTGQPLCIC HisRS1^(N6)Protein/ MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 26 HRS(1-48) Human/LKAQLGPD 1-48

For some HRS polypeptides, about or at least about 20-40, 20-45, 20-50,20-55, or 20-60, 20-65, or 20-67 contiguous or non-contiguous aminoacids of the HRS polypeptide are from amino acids 1-67 of SEQ ID NO:1.In particular embodiments, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, or 67 contiguous or non-contiguous amino acids of the HRSpolypeptide are from amino acids 1-67 of SEQ ID NO:1. The HRSpolypeptide may comprise one or more of a WHEP domain, an aminoacylationdomain, an anticodon binding domain, or any combination thereof. Inparticular embodiments, the HRS polypeptide lacks a functionalaminoacylation domain. In some embodiments, the polypeptide consistsessentially of the WHEP domain from human HRS. Without wishing to bebound by any one theory, the unique orientation, or conformation, of theWHEP domain in certain HRS polypeptides may contribute to the enhancednon canonical, and/or antibody blocking activities observed in theseproteins.

Hence, in certain embodiments, the HRS polypeptide comprises, consists,or consists essentially of a human HRS WHEP domain sequence. In someembodiments, the human HRS WHEP domain sequence is defined by certainconserved residues. For example, in some aspects the HRS polypeptidecomprises, consists, or consists essentially of the human HRS WHEPdomain consensus sequence in Table D5 below.

In certain embodiments, the HRS polypeptide may comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, or all 29 amino acids of a flexible linker connectingthe minimum domain to a heterologous protein (e.g., Fc domain), orsplice variant.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolypeptides include “reference sequence,” “comparison window,”“sequence identity,” “percentage of sequence identity” and “substantialidentity.” A “reference sequence” is at least 12 but frequently 15 to 18and often at least 25 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polypeptides may each comprise (1)a sequence (i.e., only a portion of the complete polypeptides sequence)that is similar between the two polypeptides, and (2) a sequence that isdivergent between the two polypeptides, sequence comparisons between two(or more) polypeptides are typically performed by comparing sequences ofthe two polypeptides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) can be performedas follows. To determine the percent identity of two amino acidsequences, or of two nucleic acid sequences, the sequences can bealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In certain embodiments, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch,(1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used unlessotherwise specified) are a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Thepercent identity between two amino acid or nucleotide sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (1989,Cabios, 4: 11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases, forexample, to identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.

In certain embodiments, variant polypeptides differ from thecorresponding HRS reference sequences by at least 1% but less than 20%,15%, 10% or 5% of the residues. If this comparison requires alignment,the sequences should be aligned for maximum similarity. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences. The differences are, suitably, differences or changes at anon-essential residue or a conservative substitution. In certainembodiments, the molecular weight of a variant HRS polypeptide differsfrom that of the HRS reference polypeptide by about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,or more.

Also included are biologically active “fragments” of the HRS referencepolypeptides, i.e., biologically active fragments of the HRS proteinfragments. Representative biologically active fragments generallyparticipate in an interaction, e.g., an intramolecular or aninter-molecular interaction. An inter-molecular interaction can be aspecific binding interaction or an enzymatic interaction. Aninter-molecular interaction can be between a HRS polypeptide and acellular binding partner, such as a cellular receptor or other hostmolecule that participates in the non-canonical activity of the HRSpolypeptide.

A biologically active fragment of an HRS reference polypeptide can be apolypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 38, 359, 360, 361, 362, 363, 364, 365, 380,400, 450, 500, 505, or more contiguous or non-contiguous amino acids,including all integers (e.g., 101, 102, 103) and ranges (e.g., 50-100,50-150, 50-200) in between, of the amino acid sequences set forth in anyone of the HRS reference polypeptides described herein. In certainembodiments, a biologically active fragment comprises a non-canonicalactivity-related sequence, domain, or motif. In certain embodiments, theC-terminal or N-terminal region of any HRS reference polypeptide may betruncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450,500 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150,150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 or moreamino acids, including all integers and ranges in between (e.g., 101,102, 103, 104, 105), so long as the truncated HRS polypeptide retainsthe non-canonical activity of the reference polypeptide. Certainexemplary truncated HRS polypeptides and a human HRS WHEP domainconsensus sequence are shown in Table D5 below.

TABLE D5 Exemplary truncated HRS polypeptides HRS range SequenceSEQ ID NO: C-terminal truncations  1-80MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  27LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  1-79MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  28LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDV  1-78MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  29LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFD  1-77MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  30LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVF  1-76MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  31LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKV  1-75MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  32LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREK  1-74MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  33LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVRE  1-73MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  34LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVR  1-72MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  35LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAV  1-71MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  36LKAQLGPDESKQKFVLKTPKGTRDYSPRQMA  1-70MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  37LKAQLGPDESKQKFVLKTPKGTRDYSPRQM  1-69MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  38LKAQLGPDESKQKFVLKTPKGTRDYSPRQ  1-68MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  39LKAQLGPDESKQKFVLKTPKGTRDYSPR  1-67MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  40 LKAQLGPDESKQKFVLKTPKGTRDYSP 1-66 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  41LKAQLGPDESKQKFVLKTPKGTRDYS  1-65MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  42 LKAQLGPDESKQKFVLKTPKGTRDY 1-64 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  43LKAQLGPDESKQKFVLKTPKGTRD  1-63 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK 44 LKAQLGPDESKQKFVLKTPKGTR  1-62MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  45 LKAQLGPDESKQKFVLKTPKGT 1-61 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  46 LKAQLGPDESKQKFVLKTPKG 1-60 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  47 LKAQLGPDESKQKFVLKTPK 1-59 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  48 LKAQLGPDESKQKFVLKTP 1-58 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  49 LKAQLGPDESKQKFVLKT 1-57 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  50 LKAQLGPDESKQKFVLK 1-56 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  51 LKAQLGPDESKQKFVL 1-55 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  52 LKAQLGPDESKQKFV  1-54MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  53 LKAQLGPDESKQKF  1-53MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  54 LKAQLGPDESKQK  1-52MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  55 LKAQLGPDESKQ  1-51MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  56 LKAQLGPDESK  1-50MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  57 LKAQLGPDES  1-49MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  58 LKAQLGPDE  1-48MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPD  59  1-47MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGP  60  1-46MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLG  61  1-45MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQL  62  1-44MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQ  63  1-43MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  64 LKA  1-42MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  65 LK  1-41MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  66 L  1-40MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  67 N-terminal truncations 2-80 AERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  68LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  3-80ERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  69LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  4-80RAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  70LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  5-80AALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  71LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  6-80ALEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  72LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  7-80LEELVKLQGERVRGLKQQKASAELIEEEVAKLLK  73LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  8-80EELVKLQGERVRGLKQQKASAELIEEEVAKLLK  74LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  9-80ELVKLQGERVRGLKQQKASAELIEEEVAKLLK  75LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 10-80LVKLQGERVRGLKQQKASAELIEEEVAKLLK  76LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 11-80VKLQGERVRGLKQQKASAELIEEEVAKLLK  77LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 12-80KLQGERVRGLKQQKASAELIEEEVAKLLK  78LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 13-80LQGERVRGLKQQKASAELIEEEVAKLLK  79LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 14-80QGERVRGLKQQKASAELIEEEVAKLLK  80 LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI15-80 GERVRGLKQQKASAELIEEEVAKLLK  81LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 16-80 RVRGLKQQKASAELIEEEVAKLLK 82 LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 17-80VRGLKQQKASAELIEEEVAKLLK  83 LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI18-80 RGLKQQKASAELIEEEVAKLLK  84LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 19-80 GLKQQKASAELIEEEVAKLLK  85LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 20-80 LKQQKASAELIEEEVAKLLK  86LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 21-80 KQQKASAELIEEEVAKLLK  87LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 22-80 QQKASAELIEEEVAKLLK  88LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 23-80 QKASAELIEEEVAKLLK  89LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 24-80 KASAELIEEEVAKLLK  90LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 25-80 ASAELIEEEVAKLLK  91LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 26-80 SAELIEEEVAKLLK  92LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 27-80 AELIEEEVAKLLK  93LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 28-80 ELIEEEVAKLLK  94LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 29-80 LIEEEVAKLLK  95LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 30-80 IEEEVAKLLK  96LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 31-80 EEEVAKLLK  97LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 32-80EEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  98 33-80EVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI  99 34-80VAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 100 35-80AKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 101 36-80KLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 102 37-80LLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 103 38-80LKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 104 39-80KLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 105 40-80LKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVI 106 HRSX_(A)-L-X_(B)-Q-G-X-X-V-R-X-L-K-X-X-K-A-X_(C)-V-X-X-L-L-X-L-K-X_(D) 322WHEP Where consensus X is any amino acid X_(A) is 0-50 amino acidsX_(B) is about 5-7 amino acids, preferably 6 amino acidsX_(C) is about 7-9 amino acids, preferably 8 amino acidsX_(D) is 0-50 amino acids

It will be appreciated that in any of the HRS-Fc conjugates of theinvention, the N-terminal acid of the HRS polypeptide (for example, theN-terminal Met) may additionally be deleted from any of the exemplarytruncated HRS polypeptides or other HRS sequences described herein.

Typically, the biologically-active fragment has no less than about 1%,10%, 25%, or 50% of an activity of the biologically-active (i.e.,non-canonical activity) HRS reference polypeptide from which it isderived. Exemplary methods for measuring such non-canonical activitiesare described in the Examples.

In some embodiments, HRS proteins, variants, and biologically activefragments thereof, bind to one or more cellular binding partners with anaffinity of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, or150 nM. In some embodiments, the binding affinity of a HRS proteinfragment for a selected cellular binding partner, particularly a bindingpartner that participates in a non-canonical activity, can be strongerthan that of the corresponding full length HRS polypeptide or a specificalternatively spliced HRS polypeptide variant, by at least about 1.5×,2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×,30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×,700×, 800×, 900×, 1000× or more (including all integers in between).

As noted above, a HRS polypeptide may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of a HRS referencepolypeptide can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S.Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of theGene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.).

Biologically active truncated and/or variant HRS polypeptides maycontain conservative amino acid substitutions at various locations alongtheir sequence, as compared to a reference HRS amino acid residue, andsuch additional substitutions may further enhance the activity orstability of the HRS polypeptides with altered cysteine content. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, which can be generally sub-classified asfollows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH. Amino acids having an acidic side chain include glutamic acid andaspartic acid.

Basic: The residue has a positive charge due to association with H ionat physiological pH or within one or two pH units thereof (e.g.,histidine) and the residue is attracted by aqueous solution so as toseek the surface positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium at physiological pH.Amino acids having a basic side chain include arginine, lysine andhistidine.

Charged: The residues are charged at physiological pH and, therefore,include amino acids having acidic or basic side chains (i.e., glutamicacid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and theresidue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. Amino acids having a hydrophobic sidechain include tyrosine, valine, isoleucine, leucine, methionine,phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is not sufficiently repelled by aqueous solutions so that itwould seek inner positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium. Amino acids having aneutral/polar side chain include asparagine, glutamine, cysteine,histidine, serine and threonine.

This description also characterizes certain amino acids as “small” sincetheir side chains are not sufficiently large, even if polar groups arelacking, to confer hydrophobicity. With the exception of proline,“small” amino acids are those with four carbons or less when at leastone polar group is on the side chain and three carbons or less when not.Amino acids having a small side chain include glycine, serine, alanineand threonine. The gene-encoded secondary amino acid proline is aspecial case due to its known effects on the secondary conformation ofpeptide chains. The structure of proline differs from all the othernaturally-occurring amino acids in that its side chain is bonded to thenitrogen of the α-amino group, as well as the α-carbon. Several aminoacid similarity matrices are known in the art (see e.g., PAM120 matrixand PAM250 matrix as disclosed for example by Dayhoff et al., 1978, Amodel of evolutionary change in proteins). Matrices for determiningdistance relationships In M. O. Dayhoff, (ed.), Atlas of proteinsequence and structure, Vol. 5, pp. 345-358, National BiomedicalResearch Foundation, Washington D.C.; and by Gonnet et al., (Science,256: 14430-1445, 1992), however, include proline in the same group asglycine, serine, alanine and threonine. Accordingly, for the purposes ofthe present invention, proline is classified as a “small” amino acid.

The degree of attraction or repulsion required for classification aspolar or nonpolar is arbitrary and, therefore, amino acids specificallycontemplated by the invention have been classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further sub-classified as cyclic ornon-cyclic, and aromatic or non-aromatic, self-explanatoryclassifications with respect to the side-chain substituent groups of theresidues, and as small or large. The residue is considered small if itcontains a total of four carbon atoms or less, inclusive of the carboxylcarbon, provided an additional polar substituent is present; three orless if not. Small residues are, of course, always non-aromatic.Dependent on their structural properties, amino acid residues may fallin two or more classes. For the naturally-occurring protein amino acids,sub-classification according to this scheme is presented in Table A.

TABLE A Amino acid sub-classification. Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that influenceGlycine and Proline chain orientation

Conservative amino acid substitution also includes groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulphur-containing side chains is cysteineand methionine. For example, it is reasonable to expect that replacementof a leucine with an isoleucine or valine, an aspartate with aglutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the properties of the resulting variant polypeptide. Whetheran amino acid change results in a functional truncated and/or variantHRS polypeptide can readily be determined by assaying its non-canonicalactivity, as described herein. Conservative substitutions are shown inTable B under the heading of exemplary substitutions. Amino acidsubstitutions falling within the scope of the invention, are, ingeneral, accomplished by selecting substitutions that do not differsignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, (b) the charge orhydrophobicity of the molecule at the target site, (c) the bulk of theside chain, or (d) the biological function. After the substitutions areintroduced, the variants are screened for biological activity.

TABLE B Exemplary Amino Acid Substitutions. Preferred Original ResidueExemplary Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser, Ala, Leu,Val Ser, Ala Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro HisAsn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu LeuNorleu, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile,Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser SerTrp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala,Norleu Leu

Alternatively, similar amino acids for making conservative substitutionscan be grouped into three categories based on the identity of the sidechains. The first group includes glutamic acid, aspartic acid, arginine,lysine, histidine, which all have charged side chains; the second groupincludes glycine, serine, threonine, cysteine, tyrosine, glutamine,asparagine; and the third group includes leucine, isoleucine, valine,alanine, proline, phenylalanine, tryptophan, methionine, as described inZubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

The NMR structure of the human HRS WHEP domain has been determined (seeNameki et al., Accession 1X59 A). Further, the crystal structures offull-length human HRS and an internal catalytic domain deletion mutantof HRS (HRSACD) have also been determined (see Xu et al., Structure.20:1470-7, 2012; and U.S. Application No. 61/674,639). In conjunctionwith the primary amino acid sequence of HRS, these detailed physicaldescriptions of the protein provide precise insights into the rolesplayed by specific amino acids within the protein. Persons skilled inthe art can thus use this information to identify structurally-conserveddomains, linking regions, secondary structures such as alpha-helices,surface or solvent-exposed amino acids, non-exposed or internal regions,catalytic sites, and ligand-interacting surfaces, among other structuralfeatures. Such persons can then use that and other information toreadily engineer HRS variants that retain or improve the non-canonicalactivity of interest, for instance, by conserving or altering thecharacteristics of the amino acid residues within or adjacent to theseand other structural features, such as by conserving or altering thepolarity, hydropathy index, charge, size, and/or positioning (i.e.,inward, outward) of selected amino acid side chain(s) relative towild-type residues (see, e.g., Zaiwara et al., Mol Biotechnol.51:67-102, 2012; Perona and Hadd, Biochemistry. 51:8705-29, 2012; Morinet al., Trends Biotechol. 29:159-66, 2011; Collins et al., Annu. Rev.Biophys. 40:81-98, 2011; and U.S. Application No. 61/674,639).

Thus, a predicted non-essential amino acid residue in a truncated and/orvariant HRS polypeptide is typically replaced with another amino acidresidue from the same side chain family. Alternatively, mutations can beintroduced randomly along all or part of a HRS coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran activity of the parent polypeptide to identify mutants which retainthat activity. Following mutagenesis of the coding sequences, theencoded peptide can be expressed recombinantly and the activity of thepeptide can be determined. A “non-essential” amino acid residue is aresidue that can be altered from the reference sequence of an embodimentpolypeptide without abolishing or substantially altering one or more ofits non canonical activities. Suitably, the alteration does notsubstantially abolish one of these activities, for example, the activityis at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of thereference HRS sequence. An “essential” amino acid residue is a residuethat, when altered from the reference sequence of a HRS polypeptide,results in abolition of an activity of the parent molecule such thatless than 20% of the reference activity is present. For example, suchessential amino acid residues include those that are conserved in HRSpolypeptides across different species, including those sequences thatare conserved in the active binding site(s) or motif(s) of HRSpolypeptides from various sources.

Assays to determine anti-inflammatory activity, including routinemeasurements of cytokine release from in vitro cell based, and animalstudies are well established in the art (see, for example, Wittmann etal., J Vis Exp. (65):e4203. doi: 10.3791/4203, 2012; Feldman et al., MolCell. 47:585-95, 2012; Clutterbuck et al., J Proteomics. 74:704-15,2011, Giddings and Maitra, J Biomol Screen. 15:1204-10, 2010; Wijnhovenet al., Glycoconj J. 25:177-85, 2008; and Frow et al., Med Res Rev.24:276-98, 2004) and can be readily used to profile and optimizeanti-inflammatory activity. An exemplary in vivo experimental system isalso described in the accompanying Examples.

In some embodiments, HRS polypeptides may have one or more cysteinesubstitutions, where one or more naturally-occurring (non-cysteine)residues are substituted with cysteine (e.g., to alter stability, tofacilitate thiol-based conjugation of an Fc fragment, to facilitatethiol-based attachment of PEG or other molecules). In some embodiments,cysteine substitutions are near the N-terminus and/or C-terminus of theHRS polypeptide (e.g., SEQ ID NOS:1-106, 170-181, or 185-191), or othersurface exposed regions of a HRS polypeptide. Particular embodimentsinclude where one or more of residues within 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25amino acids relative to the N-terminus and/or C-terminus of any one ofSEQ ID NOS: 1-106, 170-181, or 185-191 are substituted with a cysteineresidue. In some embodiments, cysteine residues may be added to the HRSpolypeptide through the creation of N, or C-terminal fusion proteins.Such fusion proteins may be of any length, but will typically be about1-5, or about 5-10, about 10 to 20, or about 20 to 30 amino acids inlength. In some embodiments, fusion to the C-terminus is preferred.

Specific exemplary embodiments of such cysteine modified proteins areshown in Table D6, based on the HRS polypeptide HRS(1-60). This approachis directly applicable to the HRS polypeptides of Table D5, and otherHRS polypeptides described herein.

TABLE D6 SEQ ID Name Protein Sequences NO: HRS(1-60)- M CAERAALEE LVKLQGERVR GLKQQKASAE LIEEEVAKLL 179 M1MC-KLKAQLGPDE SKQKFVLKTP K HRS(1-60)- MAERAALEEL VKLQGERVRG LKQQK CSAEL IEEEVAKLLK 180 A26C- LKAQLGPDES KQKFVLKTPK HRS(1-60)-MAERAALEEL VKLQGERVRG LKQQKASAEL IEEEVAKLLK 181 C61LKAQLGPDES KQKFVLKTPK  C DNA sequences HRS(1-60)-ATGTGTGCAGAAAGAGCCGCCCTGGAAGAGTTAGTTAAGTTGCAAGGT M1MC-GAACGTGTCCGTGGTCTGAAGCAGCAGAAGGCTAGCGCGGAGCTGAT 182CGAAGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAA HRS(1-60)-ATGGCAGAACGTGCGGCATTGGAAGAATTGGTTAAACTGCAAGGTGA A26C-ACGTGTTCGTGGTCTGAAGCAGCAGAAGTGCAGCGCGGAGCTGATCGA 183AGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAA HRS(1-60)-ATGGCAGAACGTGCGGCATTGGAAGAATTGGTTAAACTGCAAGGTGA C61ACGTGTTCGTGGTCTGAAGCAGCAGAAGGCTAGCGCGGAGCTGATCGA 184AGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAATGC

In some embodiments, the HRS polypeptide can include mutants in whichthe endogenous or naturally-occurring cysteine residues have beenmutated to alternative amino acids, or deleted. In some embodiments, theinsertion or substitution of cysteine residue(s) into the HRSpolypeptide may be combined with the elimination of other surfaceexposed reactive cysteine residues. Accordingly, in some embodiments,the HRS polypeptide may comprise one or more substitutions and/ordeletions at Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379,Cys455, Cys507, and/or Cys509 (as defined by SEQ ID NO:1), for instance,to remove naturally-occurring cysteine residues.

Specific embodiments include any one of SEQ ID NOS:1-106, 170-181, or185-191, or variants or fragments thereof, having at mutation ordeletion of any one or more of Cys83, Cys174, Cys191, Cys196, Cys224,Cys235, Cys379, Cys455, or the deletion of Cys507 and Cys509, forinstance, by the deletion of the C-terminal 3 amino acids (4507-509).Exemplary mutations at these positions include for example the mutationof cysteine to serine, alanine, leucine, valine or glycine. In certainembodiments, amino acid residues for specific cysteine substitutions canbe selected from naturally-occurring substitutions that are found in HRSorthologs from other species and organisms. Exemplary substitutions ofthis type are presented in Table D7.

TABLE D7 Naturally-occurring sequence variation at positions occupied bycysteine residues in human HRS H. sapiens cysteine P. M. B. M. R. G. X.D. D. C. S. E. residue # troglodyte mulatta taurus musculus norvegicusgallus laevis rerio melanogaster elegans cerevisiae coli 83 C C C C C CC C V T L V 174 C C C C C C C C C C C L 191 C C C C C C C C C V C A/L196 C C C C C Q H Y S M V L/A 224 C C C C C C C C C S A A 235 C C C C CC C C C C S E 379 C C C C C C C V C C C A 455 C C C C C C C — C C A A507 C R C S S — — — — S/Q S/E — 509 C C C C — — — — — I I/G —

In some embodiments, the naturally-occurring cysteines selected formutagenesis are selected based on their surface exposure. Accordingly,in one aspect the cysteine residues selected for substitution areselected from Cys224, Cys235, Cys507 and Cys509. In some embodiments,the last three (C-terminal) residues of SEQ ID NO:1 are deleted so as todelete residues 507 to 509. In some embodiments, the cysteines areselected for mutation or deletion so as to eliminate an intramolecularcysteine pair, for example Cys174 and Cys191.

Specific additional examples of desired cysteine mutations/substitutions(indicated in bold underline) to reduce surface exposed cysteineresidues include those listed below in Table D8.

TABLE D8HRS polypeptides with Substitutions to Remove Surface Exposed CysteinesSEQ ID Name Protein Sequence NO: HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 185 C174AQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAK VYRRDNPAMTRGRYREFYQA DFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 186 C174VQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAK VYRRDNPAMTRGRYREFYQV DFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 187 C191AQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAE A LKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 188 C191SQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAE S LKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 189 C191VQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAE V LKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 190 C224SQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGD FLVKVNDRRILDGMFAI SGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL HRS(1-506)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESK 191 C235SQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTI S SSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPL SEQ ID Name DNA sequences NO:HRS(1-506) ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 192 C174AGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGGCTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGTGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 193 C174VGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGGTTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGTGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 194 C191AGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGGCTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 195 C191SGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGAGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 196 C191VGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGGTTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 197 C224SGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGTGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTCCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA HRS(1-506)ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAAC 198 C235SGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGTGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTCCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTA

In some embodiments, such cysteine substituted mutants are modified toengineer-in, insert, or otherwise introduce a new surface exposedcysteine residue at a defined surface exposed position, where theintroduced residue does not substantially interfere with thenon-canonical activity of the HRS polypeptide. Specific examples includefor example the insertion (or re-insertion back) of additional cysteineresidues at the N- or C-terminus of any of the reduced cysteine HRSpolypeptides described above. In some embodiments, the insertion of suchN- or C-terminal surface exposed cysteines involves the re-insertion ofthe last 1, last 2, or last 3 naturally occurring C-terminal amino acidsof the full length human HRS to a reduced cysteine variant of a HRSpolypeptide e.g., the re-insertion of all or part of the sequence CIC(Cys Ile Cys). Exemplary reduced cysteine mutants include for exampleany combination of mutations (or the deletion of) at residues Cys174,Cys191, Cys224, and Cys235, and or the deletion or substitution ofCys507 and Cys509 (based on the numbering of full length human HRS (SEQID NO:1) in any of the HRS polypeptides of SEQ ID NOS: 1-106, 170-181,or 185-191 or Tables D1, D3-D6 or D8.

For some types of site-specific conjugation or attachment toheterologous molecules such as Fc regions or PEG or other heterologousmolecules, HRS polypeptides may have one or more glutaminesubstitutions, where one or more naturally-occurring (non-glutamine)residues are substituted with glutamine, for example, to facilitatetransglutaminase-catalyzed attachment of the molecule(s) to theglutamine's amide group. In some embodiments, glutamine substitutionsare introduced near the N-terminus and/or C-terminus of the HRSpolypeptide (e.g., SEQ ID NOS:1-106, 170-181, or 185-191 or the HRSpolypeptides of Tables D1, D3-D6 or D8). Particular embodiments includewhere one or more of residues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acidsrelative to the N-terminus and/or C-terminus of any one of SEQ IDNOS:1-106, 170-181, or 185-191 are substituted with a glutamine residue.These and related HRS polypeptides can also include substitutions (e.g.,conservative substitutions) to remove any naturally-occurring glutamineresidues, if desired, and thereby regulate the degree of site-specificconjugation or attachment.

For certain types of site-specific conjugation or attachment toheterologous molecules such as Fc regions or PEG or other heterologousmolecules, HRS polypeptides may have one or more lysine substitutions,where one or more naturally-occurring (non-lysine) residues aresubstituted with lysine, for example, to facilitate acylation oralkylation-based attachment of molecule(s) to the lysine's amino group.These methods also typically result in attachment of molecule(s) to theN-terminal residue. In some embodiments, lysine substations are near theN-terminus and/or C-terminus of the HRS polypeptide (e.g., SEQ IDNOS:1-106, 170-181, or 185-191 or the HRS polypeptides of Tables D1,D3-D6 or D8). Particular embodiments include where one or more ofresidues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids to the N-terminusand/or C-terminus of any one of SEQ ID NOS:1-106, 170-181, or 185-191(or the HRS polypeptides of Tables D1, D3-D6 or D8) are substituted witha lysine residue. These and related HRS polypeptides can also includesubstitutions (e.g., conservative substitutions) to remove anynaturally-occurring lysine residues, if desired, and thereby regulatethe degree of site-specific conjugation or attachment.

Site-specific conjugation to HRS polypeptides may also be performed bysubstituting one or more solvent accessible surface amino acids of a HRSpolypeptide. For example, suitable solvent accessible amino acids may bedetermined based on the predicted solvent accessibility using theSPIDDER server (http://sppider.cchmc.org/) using the published crystalstructure of an exemplary HRS polypeptide (see Xu et al., Structure.20:1470-7, 2012; and U.S. Application No. 61/674,639). Based on thisanalysis several amino acids on the surface may potentially be used asmutation sites to introduce functional groups suitable for conjugationor attachment. The surface accessibility score of amino acids based onthe crystal structure can be calculated, where the higher scoresrepresent better accessibility. In particular embodiments, higher scores(for example, >40) are preferred. Accordingly in some embodiments anamino acid position have a surface accessibility score of greater than40 may used to introduce a cysteine, lysine, glutamine, or othernon-naturally-occurring amino acid.

In particular embodiments, a solvent accessible surface amino acid isselected from the group consisting of: alanine, glycine, and serine, andcan be substituted with naturally occurring amino acids including, butnot limited to, cysteine, glutamine, or lysine, or a non-naturallyoccurring amino acid that is optimized for site specific conjugation orattachment.

In various embodiments, the present invention contemplates site-specificconjugation or attachment at any amino acid position in a HRSpolypeptide by virtue of substituting a non-naturally-occurring aminoacid comprising a functional group that will form a covalent bond withthe functional group attached to a heterologous molecules such as an Fcregion or PEG or other heterologous molecule. Non-natural amino acidscan be inserted or substituted at, for example, one or more of residueswithin 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 amino acids relative to the N-terminusand/or C-terminus of any one of SEQ ID NOS:1-106, 170-181, or 185-191(or the HRS polypeptides of Tables D1, D3-D6 or D8); at the N-terminusand/or C-terminus of any one of SEQ ID NOS:1-106, 170-181, or 185-191(or the HRS polypeptides of Tables D1, D3-D6 or D8); or a solventaccessible surface amino acid residue as described herein.

In particular embodiments, non-naturally occurring amino acids include,without limitation, any amino acid, modified amino acid, or amino acidanalogue other than selenocysteine and the following twenty geneticallyencoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by the following formula:

A non-natural amino acid is typically any structure having the foregoingformula wherein the R group is any substituent other than one used inthe twenty natural amino acids. See, e.g., biochemistry texts such asBiochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York,for structures of the twenty natural amino acids. Note that thenon-natural amino acids disclosed herein may be naturally occurringcompounds other than the twenty alpha-amino acids above. Because thenon-natural amino acids disclosed herein typically differ from thenatural amino acids in side chain only, the non-natural amino acids formamide bonds with other amino acids, e.g., natural or non-natural, in thesame manner in which they are formed in naturally occurring proteins.However, the non-natural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R inforegoing formula optionally comprises an alkyl-, aryl-, aryl halide,vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro,halide, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynyl, ether, thio ether, epoxide, sulfone,boronic acid, boronate ester, borane, phenylboronic acid, thiol,seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine,heterocyclic-, pyridyl, naphthyl, benzophenone, a constrained ring suchas a cyclooctyne, thio ester, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amino, carboxylic acid, alpha-keto carboxylic acid, alphaor beta unsaturated acids and amides, glyoxyl amide, or organosilanegroup, or the like or any combination thereof.

Specific examples of unnatural amino acids include, but are not limitedto, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, atri-O-acetyl-GalNAc-α-threonine, an α-GalNAc-L-threonine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, those listedbelow, or elsewhere herein, and the like.

Accordingly, one may select a non-naturally occurring amino acidcomprising a functional group that forms a covalent bond with anypreferred functional group of a desired molecule (e.g., Fc region, PEG).Non-natural amino acids, once selected, can either be purchased fromvendors, or chemically synthesized. Any number of non-natural aminoacids may be incorporated into the target molecule and may varyaccording to the number of desired molecules that are to be attached.The molecules may be attached to all or only some of the non-naturalamino acids. Further, the same or different non-natural amino acids maybe incorporated into a HRS polypeptide, depending on the desiredoutcome. In certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore non-natural amino acids are incorporated into a HRS polypeptide anyor all of which may be conjugated to a molecule comprising a desiredfunctional group.

In certain aspects, the use of non-natural amino acids can be utilizedto modify (e.g., increase) a selected non-canonical activity of a HRSpolypeptide, or to alter the in vivo or in vitro half-life of theprotein. Non-natural amino acids can also be used to facilitate(selective) chemical modifications (e.g., pegylation) of a HRS protein,as described elsewhere herein. For instance, certain non-natural aminoacids allow selective attachment of polymers such as an Fc region or PEGto a given protein, and thereby improve their pharmacokineticproperties.

Specific examples of amino acid analogs and mimetics can be founddescribed in, for example, Roberts and Vellaccio, The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341,Academic Press, Inc., New York, N.Y. (1983), the entire volume of whichis incorporated herein by reference. Other examples include peralkylatedamino acids, particularly permethylated amino acids. See, for example,Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, JohnWiley & Sons Inc., New York, N.Y. (1997), the entire book of which isincorporated herein by reference. Yet other examples include amino acidswhose amide portion (and, therefore, the amide backbone of the resultingpeptide) has been replaced, for example, by a sugar ring, steroid,benzodiazepine or carbo cycle. See, for instance, Burger's MedicinalChemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620,John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of whichis incorporated herein by reference. Methods for synthesizing peptides,polypeptides, peptidomimetics and proteins are well known in the art(see, for example, U.S. Pat. No. 5,420,109; M. Bodanzsky, Principles ofPeptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York,N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid PhasePeptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984),each of which is incorporated herein by reference). Accordingly, the HRSpolypeptides of the present invention may be composed of naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics.

Polynucleotides

Certain embodiments relate to polynucleotides that encode a HRSpolypeptide or a HRS-Fc fusion protein. Also included arepolynucleotides that encode any one or more of the Fc regions describedherein, alone or in combination with a HRS coding sequence. Among otheruses, these embodiments may be utilized to recombinantly produce adesired HRS, Fc region, or HRS-Fc polypeptide or variant thereof, or toexpress the HRS, Fc region, or HRS-Fc polypeptide in a selected cell orsubject. It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a HRS polypeptide HRS-Fc fusion proteinas described herein. Some of these polynucleotides may bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention, for examplepolynucleotides that are optimized for human, yeast or bacterial codonselection.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an HRS-Fc fusion polypeptide or a portion thereof)or may comprise a variant, or a biological functional equivalent of sucha sequence. Polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions, as furtherdescribed below, preferably such that the activity of the encodedpolypeptide is not substantially diminished relative to the unmodifiedpolypeptide.

In additional embodiments, the present invention provides isolatedpolynucleotides comprising various lengths of contiguous stretches ofsequence identical to or complementary to a HRS polypeptide or HRS-Fcfusion protein, wherein the isolated polynucleotides encode a truncatedHRS polypeptide as described herein

Therefore, multiple polynucleotides can encode the HRS polypeptides, Fcregions, and fusion proteins of the invention. Moreover, thepolynucleotide sequence can be manipulated for various reasons. Examplesinclude but are not limited to the incorporation of preferred codons toenhance the expression of the polynucleotide in various organisms (seegenerally Nakamura et al., Nuc. Acid. Res. 28:292, 2000). In addition,silent mutations can be incorporated in order to introduce, or eliminaterestriction sites, decrease the density of CpG dinucleotide motifs (seefor example, Kameda et al., Biochem. Biophys. Res. Commun.349:1269-1277, 2006) or reduce the ability of single stranded sequencesto form stem-loop structures: (see, e.g., Zuker M., Nucl. Acid Res.31:3406-3415, 2003). In addition, mammalian expression can be furtheroptimized by including a Kozak consensus sequence (i.e.,(a/g)cc(a/g)ccATGg) (SEQ ID NO:199) at the start codon. Kozak consensussequences useful for this purpose are known in the art (Mantyh et al.,PNAS 92: 2662-2666, 1995; Mantyh et al., Prot. Exp. & Purif. 6:124,1995). Exemplary wild type and codon optimized versions of various HRSpolypeptides are provided in Table D9 below.

TABLE D9 Codon Optimized DNA Sequences Amino Acid Residue Range ofSEQ ID SEQ ID Name NO: 1 Nucleic acid sequence NO: Wild type   1-509ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCA 111 (Full lengthGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCG HisRS)CCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGGGCACAAGAGACTATAGTCCCCGGCAGATGGCAGTTCGCGAGAAGGTGTTTGACGTAATCATCCGTTGCTTCAAGCGCCACGGTGCAGAAGTCATTGATACACCTGTATTTGAACTAAAGGAAACACTGATGGGAAAGTATGGGGAAGACTCCAAGCTTATCTATGACCTGAAGGACCAGGGCGGGGAGCTCCTGTCCCTTCGCTATGACCTCACTGTTCCTTTTGCTCGGTATTTGGCAATGAATAAACTGACCAACATTAAACGCTACCACATAGCAAAGGTATATCGGCGGGATAACCCAGCCATGACCCGTGGCCGATACCGGGAATTCTACCAGTGTGATTTTGACATTGCTGGGAACTTTGATCCCATGATCCCTGATGCAGAGTGCCTGAAGATCATGTGCGAGATCCTGAGTTCACTTCAGATAGGCGACTTCCTGGTCAAGGTAAACGATCGACGCATTCTAGATGGGATGTTTGCTATCTGTGGTGTTTCTGACAGCAAGTTCCGTACCATCTGCTCCTCAGTAGACAAGCTGGACAAGGTGTCCTGGGAAGAGGTGAAGAATGAGATGGTGGGAGAGAAGGGCCTTGCACCTGAGGTGGCTGACCGCATTGGGGACTATGTCCAGCAACATGGTGGGGTATCCCTGGTGGAACAGCTGCTCCAGGATCCTAAACTATCCCAAAACAAGCAGGCCTTGGAGGGCCTGGGAGACCTGAAGTTGCTCTTTGAGTACCTGACCCTATTTGGCATTGATGACAAAATCTCCTTTGACCTGAGCCTTGCTCGAGGGCTGGATTACTACACTGGGGTGATCTATGAGGCAGTGCTGCTACAGACCCCAGCCCAGGCAGGGGAAGAGCCCCTGGGTGTGGGCAGTGTGGCTGCTGGAGGACGCTATGATGGGCTAGTGGGCATGTTCGACCCCAAAGGGCGCAAGGTGCCATGTGTGGGGCTCAGCATTGGGGTGGAGCGGATTTTCTCCATCGTGGAACAGAGACTAGAGGCTTTGGAGGAGAAGATACGGACCACGGAGACACAGGTGCTTGTGGCATCTGCACAGAAGAAGCTGCTAGAGGAAAGACTAAAGCTTGTCTCAGAACTGTGGGATGCTGGGATCAAGGCTGAGCTGCTGTACAAGAAGAACCCAAAGCTACTGAACCAGTTACAGTACTGTGAGGAGGCAGGCATCCCACTGGTGGCTATCATCGGCGAGCAGGAACTCAAGGATGGGGTCATCAAGCTCCGTTCAGTGACGAGCAGGGAAGAGGTGGATGTCCGAAGAGAAGACCTTGTGGAGGAAATCAAAAGGAGAACAG GCCAGCCCCTCTGCATCTGCHisRS1^(N1)   1-141 ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 112AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACCCGTGACTATTCTCCTCGTCAAATGGCCGTCCGTGAAAAAGTGTTCGACGTGATCATTCGCTGCTTTAAACGCCATGGTGCCGAAGTGATTGATACCCCGGTGTTTGAGCTGAAAGAGACACTGATGGGCAAATATGGTGAGGACAGCAAACTGATTTATGACCTGAAAGATCAGGGTGGTGAACTGCTGAGTCTGCGCTATGATCTGACAGTTCCGTTTGCCCGTTATCTG GCAATG HisRS1^(N2)   1-408ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 113AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACCCGTGACTATTCTCCTCGTCAAATGGCCGTCCGTGAAAAAGTGTTCGACGTGATCATTCGCTGCTTTAAACGCCATGGTGCCGAAGTGATTGATACCCCGGTGTTTGAGCTGAAAGAGACACTGATGGGCAAATATGGTGAGGACAGCAAACTGATTTATGACCTGAAAGATCAGGGTGGTGAACTGCTGAGTCTGCGCTATGATCTGACAGTTCCGTTTGCCCGTTATCTGGCAATGAATAAACTGACCAACATTAAACGCTATCACATTGCTAAAGTCTATCGCCGTGACAATCCTGCTATGACCCGTGGTCGTTATCGTGAGTTCTATCAGTGTGACTTCGATATTGCCGGCAACTTTGATCCGATGATCCCGGATGCTGAATGCCTGAAAATCATGTGTGAGATCCTGAGCAGTCTGCAGATTGGCGATTTCCTGGTGAAAGTCAACGATCGCCGTATTCTGGATGGCATGTTCGCCATCTGTGGTGTTAGCGACTCCAAATTCCGTACCATCTGTAGTAGTGTGGACAAACTGGATAAAGTGAGCTGGGAGGAGGTGAAAAACGAAATGGTGGGCGAGAAAGGTCTGGCTCCTGAAGTGGCTGACCGTATTGGTGATTATGTCCAGCAGCACGGTGGAGTATCACTGGTTGAGCAACTGCTGCAAGACCCTAAACTGAGTCAGAATAAACAGGCCCTGGAGGGACTGGGAGATCTGAAACTGCTGTTCGAGTATCTGACCCTGTTCGGTATCGATGACAAAATCTCCTTTGACCTGTCACTGGCTCGTGGACTGGACTATTATACCGGCGTGATCTATGAAGCTGTACTGCTGCAAACTCCAGCACAAGCAGGTGAAGAGCCTCTGGGTGTGGGTAGTGTAGCCGCTGGGGGACGTTATGATGGACTGGTGGGGATGTTCGACCCTAAAGGCCGTAAAGTTCCGTGTGTGGGTCTGAGTATCGGTGTTGAGCGTATCTTTTCCATCGTCGAGCAACGTCTGGAAGCACTGGAGGAAAAAATCCGTACGA CCGAA HisRS1^(N3)   1-113ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 114AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACCCGTGACTATTCTCCTCGTCAAATGGCCGTCCGTGAAAAAGTGTTCGACGTGATCATTCGCTGCTTTAAACGCCATGGTGCCGAAGTGATTGATACCCCGGTGTTTGAGCTGAAAGAGACACTGATGGGCAAATATGGTGAGGACAG CAAACTG HisRS1^(N4)   1-60ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 115AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTC CTGAAAACTCCGAAG HisRS1^(N8)  1-506 ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCA 116GGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGGGCACAAGAGACTATAGTCCCCGGCAGATGGCAGTTCGCGAGAAGGTGTTTGACGTAATCATCCGTTGCTTCAAGCGCCACGGTGCAGAAGTCATTGATACACCTGTATTTGAACTAAAGGAAACACTGATGGGAAAGTATGGGGAAGACTCCAAGCTTATCTATGACCTGAAGGACCAGGGCGGGGAGCTCCTGTCCCTTCGCTATGACCTCACTGTTCCTTTTGCTCGGTATTTGGCAATGAATAAACTGACCAACATTAAACGCTACCACATAGCAAAGGTATATCGGCGGGATAACCCAGCCATGACCCGTGGCCGATACCGGGAATTCTACCAGTGTGATTTTGACATTGCTGGGAACTTTGATCCCATGATCCCTGATGCAGAGTGCCTGAAGATCATGTGCGAGATCCTGAGTTCACTTCAGATAGGCGACTTCCTGGTCAAGGTAAACGATCGACGCATTCTAGATGGGATGTTTGCTATCTGTGGTGTTTCTGACAGCAAGTTCCGTACCATCTGCTCCTCAGTAGACAAGCTGGACAAGGTGTCCTGGGAAGAGGTGAAGAATGAGATGGTGGGAGAGAAGGGCCTTGCACCTGAGGTGGCTGACCGCATTGGGGACTATGTCCAGCAACATGGTGGGGTATCCCTGGTGGAACAGCTGCTCCAGGATCCTAAACTATCCCAAAACAAGCAGGCCTTGGAGGGCCTGGGAGACCTGAAGTTGCTCTTTGAGTACCTGACCCTATTTGGCATTGATGACAAAATCTCCTTTGACCTGAGCCTTGCTCGAGGGCTGGATTACTACACTGGGGTGATCTATGAGGCAGTGCTGCTACAGACCCCAGCCCAGGCAGGGGAAGAGCCCCTGGGTGTGGGCAGTGTGGCTGCTGGAGGACGCTATGATGGGCTAGTGGGCATGTTCGACCCCAAAGGGCGCAAGGTGCCATGTGTGGGGCTCAGCATTGGGGTGGAGCGGATTTTCTCCATCGTGGAACAGAGACTAGAGGCTTTGGAGGAGAAGATACGGACCACGGAGACACAGGTGCTTGTGGCATCTGCACAGAAGAAGCTGCTAGAGGAAAGACTAAAGCTTGTCTCAGAACTGTGGGATGCTGGGATCAAGGCTGAGCTGCTGTACAAGAAGAACCCAAAGCTACTGAACCAGTTACAGTACTGTGAGGAGGCAGGCATCCCACTGGTGGCTATCATCGGCGAGCAGGAACTCAAGGATGGGGTCATCAAGCTCCGTTCAGTGACGAGCAGGGAAGAGGTGGATGTCCGAAGAGAAGACCTTGTGGAGGAAATCAAAAGGAGAACAG GCCAGCCCCTC HisRS1^(N6)   1-48ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 117AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTG AAAGCACAGCTGGGTCCTGATHisRS1^(I1) 191-333 TGCCTGAAAATCATGTGTGAGATCCTGAGTAGTCTGCAAATT 118GGCGACTTTCTGGTCAAAGTGAACGATCGCCGTATTCTGGATGGCATGTTCGCCATCTGTGGTGTTAGCGACTCCAAATTCCGTACAATCTGTAGCAGCGTGGACAAACTGGATAAAGTGTCCTGGGAAGAGGTGAAAAACGAAATGGTGGGTGAAAAAGGTCTGGCTCCGGAGGTTGCTGACCGTATCGGTGATTATGTTCAGCAGCACGGCGGTGTTAGTCTGGTTGAACAACTGCTGCAAGACCCGAAACTGTCTCAGAACAAACAGGCCCTGGAAGGACTGGGAGATCTGAAACTGCTGTTCGAGTATCTGACGCTGTTCGGCATTGATGACAAAATTTCTTTCGACCTGTCACTGGCACGTGGACTGGACTATT ATACCGGT HisRS1^(C1) 405-509CGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAA 119AAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTACC GGTCAACCTCTGTGTATTTGCHisRS1^(N5)   1-243 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 12027 aa AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACCCGTGACTATTCTCCTCGTCAAATGGCCGTCCGTGAAAAAGTGTTCGACGTGATCATTCGCTGCTTTAAACGCCATGGTGCCGAAGTGATTGATACCCCGGTGTTTGAGCTGAAAGAGACACTGATGGGCAAATATGGTGAGGACAGCAAACTGATCTATGACCTGAAAGACCAAGGCGGTGAACTGCTGTCCCTGCGTTATGATCTGACTGTGCCGTTTGCCCGTTATCTGGCCATGAATAAACTGACGAACATTAAACGCTATCACATTGCCAAAGTGTATCGCCGTGACAATCCTGCTATGACTCGTGGACGTTATCGTGAATTCTATCAGTGTGACTTCGATATTGCCGGCAACTTCGACCCTATGATTCCGGATGCTGAATGCCTGAAAATCATGTGTGAGATCCTGAGCAGCCTGCAAATTGGTGACTTCCTGGTGAAAGTGAATGACCGTCGTATCCTGGATGGCATGTTTGCCATTTGTGGTGTGAGCGATTCCAAATTCCGTACCATCTGTAGTAGTGTGGACAAACTGGATAAAGTGGGCTATCCGTGGTGGAACTCTTGTAGCCGTATTCTGAACTATCCTAAAACCAGCCGCCCGTGGCGT GCTTGGGAAACT HisRS1^(C2)  1-60 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 121 175-509AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGACTTCGATATTGCCGGGAATTTTGACCCTATGATCCCTGATGCCGAATGTCTGAAAATCATGTGTGAGATCCTGAGCAGTCTGCAGATTGGTGACTTCCTGGTGAAAGTGAACGATCGCCGTATTCTGGATGGAATGTTTGCCATTTGTGGCGTGTCTGACAGCAAATTTCGTACGATCTGTAGCAGCGTGGATAAACTGGATAAAGTGAGCTGGGAGGAGGTGAAAAATGAGATGGTGGGCGAAAAAGGTCTGGCACCTGAAGTGGCTGACCGTATCGGTGATTATGTTCAGCAACATGGCGGTGTTTCTCTGGTCGAACAGCTGCTGCAAGACCCAAAACTGAGCCAGAACAAACAGGCACTGGAAGGACTGGGTGATCTGAAACTGCTGTTTGAGTATCTGACGCTGTTTGGCATCGATGACAAAATCTCGTTTGACCTGAGCCTGGCACGTGGTCTGGATTATTATACCGGCGTGATCTATGAAGCCGTCCTGCTGCAAACTCCAGCACAAGCAGGTGAAGAACCTCTGGGTGTTGGTAGTGTAGCGGCAGGCGGACGTTATGATGGACTGGTGGGGATGTTTGATCCGAAAGGCCGTAAAGTTCCGTGTGTCGGTCTGAGTATCGGGGTTGAGCGTATCTTTAGCATTGTGGAGCAACGTCTGGAAGCTCTGGAGGAAAAAATCCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTACCGGTCAACCTC TGTGTATTTGC HisRS1^(C3)  1-60 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 122 211-509AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGTGAATGATCGCCGTATCCTGGATGGCATGTTTGCCATTTGTGGTGTGAGCGACTCGAAATTCCGTACGATTTGCTCTAGCGTCGATAAACTGGACAAAGTGTCCTGGGAAGAGGTGAAAAACGAGATGGTGGGTGAGAAAGGTCTGGCTCCTGAAGTTGCCGACCGTATTGGTGATTATGTTCAGCAGCATGGCGGTGTTTCACTGGTTGAACAACTGCTGCAAGACCCGAAACTGTCTCAGAATAAACAGGCGCTGGAAGGACTGGGAGATCTGAAACTGCTGTTTGAGTATCTGACCCTGTTCGGCATTGATGACAAAATCAGCTTCGACCTGAGCCTGGCACGTGGTCTGGATTATTATACCGGCGTGATCTATGAAGCCGTTCTGCTGCAGACACCAGCACAAGCAGGCGAAGAACCTCTGGGTGTTGGTTCTGTGGCAGCCGGTGGTCGTTATGATGGACTGGTAGGCATGTTCGATCCGAAAGGCCGTAAAGTTCCGTGTGTGGGACTGAGTATCGGTGTTGAGCGTATCTTTAGCATCGTGGAACAACGTCTGGAAGCGCTGGAGGAGAAAATTCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAAC GTCGTACCGGTCAACCTCTGTGTATTTGCHisRS1^(C4)   1-100 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 123211-509 AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACTCGTGATTATAGCCCTCGCCAGATGGCTGTCCGTGAAAAAGTGTTCGATGTGATCATTCGCTGCTTCAAACGTCATGGTGCCGAAGTCATTGATACCCCGGTGTTCGAGCTGAAAGTGAACGATCGCCGTATTCTGGATGGCATGTTCGCCATTTGTGGTGTTAGCGATAGCAAATTCCGTACAATCTGCTCTAGCGTGGACAAACTGGACAAAGTGAGCTGGGAAGAGGTGAAAAACGAGATGGTGGGTGAGAAAGGCCTGGCTCCTGAAGTTGCCGACCGTATCGGAGATTATGTTCAGCAGCATGGCGGAGTTTCACTGGTTGAACAACTGCTGCAAGACCCGAAACTGTCTCAGAACAAACAGGCACTGGAAGGTCTGGGAGATCTGAAACTGCTGTTCGAGTATCTGACGCTGTTCGGTATTGACGACAAAATTTCCTTCGACCTGTCGCTGGCACGTGGTCTGGATTATTATACAGGCGTGATCTATGAGGCTGTACTGCTGCAGACACCAGCACAAGCAGGTGAAGAGCCTCTGGGTGTTGGTTCAGTTGCTGCCGGTGGACGTTATGACGGACTGGTAGGGATGTTTGACCCAAAAGGCCGTAAAGTCCCGTGTGTAGGACTGTCTATTGGCGTTGAGCGTATCTTTAGCATCGTGGAGCAACGTCTGGAAGCTCTGGAGGAGAAAATCCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTAC CGGTCAACCTCTGTGTATTTGCHisRS1^(C5)   1-174 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 124211-509 AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACTCGTGATTATAGCCCTCGCCAGATGGCTGTCCGTGAAAAAGTGTTCGATGTGATCATTCGCTGCTTCAAACGTCATGGTGCCGAAGTCATTGATACCCCGGTGTTCGAGCTGAAAGAAACCCTGATGGGCAAATATGGGGAAGATTCCAAACTGATCTATGACCTGAAAGACCAGGGAGGTGAACTGCTGTCTCTGCGCTATGACCTGACTGTTCCTTTTGCTCGCTATCTGGCCATGAATAAACTGACCAACATCAAACGCTATCATATCGCCAAAGTGTATCGCCGTGACAATCCAGCAATGACCCGTGGTCGTTATCGTGAATTTTATCAGTGTGTGAACGATCGCCGTATTCTGGACGGCATGTTCGCCATTTGTGGTGTGTCTGACTCCAAATTTCGTACGATCTGCTCAAGCGTGGACAAACTGGACAAAGTGAGCTGGGAAGAGGTGAAAAACGAGATGGTGGGTGAGAAAGGCCTGGCTCCTGAAGTTGCCGACCGTATCGGAGATTATGTTCAGCAGCATGGCGGAGTTTCACTGGTTGAACAACTGCTGCAAGACCCGAAACTGTCACAGAACAAACAGGCACTGGAAGGTCTGGGGGATCTGAAACTGCTGTTCGAGTATCTGACGCTGTTCGGTATTGACGACAAAATCAGCTTCGATCTGAGCCTGGCACGTGGTCTGGACTATTATACCGGCGTGATTTATGAAGCCGTTCTGCTGCAGACTCCAGCACAAGCAGGTGAAGAGCCTCTGGGTGTTGGAAGTGTGGCAGCCGGTGGCCGTTATGATGGTCTGGTTGGCATGTTTGACCCGAAAGGCCGTAAAGTCCCGTGTGTAGGACTGTCTATCGGCGTGGAGCGTATTTTTAGCATCGTGGAACAACGCCTGGAAGCTCTGGAAGAGAAAATCCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTACCGGTCAACCTCTGTGTATTTGC HisRS1^(C6)   1-60 +ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 125 101-509AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGAAACCCTGATGGGCAAATATGGCGAAGATTCCAAACTGATCTATGACCTGAAAGACCAAGGCGGTGAACTGCTGTCCCTGCGTTATGACCTGACTGTTCCGTTTGCTCGTTATCTGGCCATGAATAAACTGACCAACATTAAACGCTATCACATTGCCAAAGTGTATCGCCGTGACAATCCTGCTATGACTCGTGGACGTTATCGTGAATTCTATCAGTGTGACTTCGATATTGCCGGCAACTTCGACCCTATGATTCCGGATGCTGAATGCCTGAAAATCATGTGTGAGATCCTGAGCAGCCTGCAAATTGGTGACTTCCTGGTGAAAGTGAATGACCGTCGTATCCTGGATGGCATGTTCGCCATTTGTGGTGTTAGCGATTCCAAATTCCGTACCATCTGTAGTAGTGTGGACAAACTGGATAAAGTGAGCTGGGAAGAGGTGAAAAACGAAATGGTGGGCGAAAAAGGTCTGGCACCTGAGGTTGCTGATCGTATCGGTGACTATGTCCAGCAGCATGGAGGTGTTTCACTGGTTGAGCAACTGCTGCAAGATCCGAAACTGTCTCAGAACAAACAGGCCCTGGAAGGACTGGGTGATCTGAAACTGCTGTTCGAGTATCTGACGCTGTTCGGTATTGATGACAAAATCTCGTTCGACCTGTCTCTGGCTCGTGGACTGGATTATTATACGGGCGTAATCTATGAAGCTGTCCTGCTGCAGACACCAGCACAAGCAGGTGAAGAGCCTCTGGGTGTTGGAAGTGTTGCTGCCGGTGGTCGCTATGACGGACTGGTTGGCATGTTCGATCCGAAAGGCCGTAAAGTTCCGTGTGTAGGACTGAGCATTGGCGTTGAGCGTATCTTTTCCATCGTTGAGCAACGTCTGGAAGCACTGGAAGAGAAAATCCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTACCG GTCAACCTCTGTGTATTTGCHisRS1^(C7)   1-100 + ATGGCAGAACGTGCCGCCCTGGAAGAGCTGGTAAAACTGCA 126175-509 AGGCGAGCGTGTTCGTGGTCTGAAACAGCAGAAAGCAAGCGCTGAACTGATCGAAGAAGAAGTGGCGAAACTGCTGAAACTGAAAGCACAGCTGGGTCCTGATGAATCAAAACAAAAATTCGTCCTGAAAACTCCGAAAGGAACTCGTGATTATAGCCCTCGCCAGATGGCTGTCCGTGAAAAAGTGTTCGATGTGATCATTCGCTGCTTCAAACGTCATGGTGCCGAAGTCATTGATACCCCGGTGTTCGAGCTGAAAGATTTCGATATTGCCGGCAACTTTGATCCGATGATTCCGGATGCTGAGTGTCTGAAAATCATGTGTGAGATCCTGAGTAGTCTGCAGATTGGGGATTTCCTGGTGAAAGTGAACGATCGCCGTATTCTGGACGGCATGTTTGCCATTTGTGGCGTTAGCGATAGCAAATTCCGTACGATCTGTAGCAGTGTGGACAAACTGGATAAAGTCTCTTGGGAAGAGGTCAAAAACGAGATGGTTGGTGAGAAAGGCCTGGCTCCTGAAGTGGCTGACCGTATTGGTGATTATGTCCAGCAGCATGGTGGTGTTTCACTGGTTGAACAACTGCTGCAAGACCCGAAACTGTCTCAGAACAAACAGGCACTGGAAGGTCTGGGTGATCTGAAACTGCTGTTCGAGTATCTGACGCTGTTCGGTATTGACGACAAAATTTCCTTCGACCTGTCACTGGCACGTGGTCTGGATTATTATACAGGCGTAATCTATGAGGCTGTACTGCTGCAAACTCCAGCACAAGCAGGTGAAGAACCTCTGGGAGTTGGTAGTGTAGCGGCAGGGGGTCGTTATGATGGGCTGGTCGGGATGTTCGATCCAAAAGGCCGTAAAGTCCCGTGTGTTGGTCTGTCTATTGGCGTTGAGCGTATCTTCTCCATCGTGGAGCAACGTCTGGAAGCTCTGGAAGAAAAAATCCGTACCACCGAAACCCAAGTTCTGGTTGCCTCAGCTCAGAAAAAACTGCTGGAAGAACGCCTGAAACTGGTTAGCGAACTGTGGGATGCTGGCATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAGGAAGCGGGTATTCCTCTGGTGGCCATTATCGGAGAACAGGAACTGAAAGACGGCGTTATTAAACTGCGTAGCGTGACCTCTCGTGAAGAAGTTGACGTTCGCCGTGAAGATCTGGTCGAGGAAATCAAACGTCGTACCGGTCAACCTCTGTGTAT TTGC HisRS1^(C10) 369-509ATGTTCGACCCAAAAGGCCGTAAAGTTCCGTGTGTAGGGCTG 127TCTATCGGTGTTGAGCGTATCTTCTCCATCGTTGAGCAGCGTCTGGAAGCACTGGAGGAAAAAATCCGTACGACCGAGACTCAAGTCCTGGTTGCTAGTGCCCAGAAAAAACTGCTGGAAGAGCGCCTGAAACTGGTTAGTGAGCTGTGGGATGCCGGTATTAAAGCCGAACTGCTGTATAAAAAAAACCCGAAACTGCTGAATCAGCTGCAGTATTGTGAAGAAGCGGGCATTCCGCTGGTAGCGATTATCGGGGAACAAGAACTGAAAGATGGCGTGATCAAACTGCGTAGCGTTACAAGCCGTGAGGAAGTGGACGTCCGCCGTGAGGATCTGGTTGAAGAGATTAAACGCCGTACAGGTCAGCCTCTGTGTAT TTGC

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide of the present invention, and a polynucleotidemay, but need not, be linked to other molecules and/or supportmaterials. Hence, the polynucleotides of the present invention,regardless of the length of the coding sequence itself, may be combinedwith other DNA or RNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably.

It is therefore contemplated that a polynucleotide fragment of almostany length may be employed; with the total length preferably beinglimited by the ease of preparation and use in the intended recombinantDNA protocol. Included are polynucleotides of about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,220, 240, 260, 270, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,3000 or more (including all integers in between) bases in length,including any portion or fragment (e.g., greater than about 6, 7, 8, 9,or 10 nucleotides in length) of a HRS reference polynucleotide (e.g.,base number X-Y, in which X is about 1-3000 or more and Y is about10-3000 or more), or its complement.

Embodiments of the present invention also include “variants” of the HRSreference polynucleotide sequences. Polynucleotide “variants” maycontain one or more substitutions, additions, deletions and/orinsertions in relation to a reference polynucleotide. Generally,variants of a HRS reference polynucleotide sequence may have at leastabout 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%,80%, 85%, desirably about 90% to 95% or more, and more suitably about98% or more sequence identity to that particular nucleotide sequence(Such as for example, SEQ ID NOS:111-127, 182-184, 192-198; see also theExamples) as determined by sequence alignment programs describedelsewhere herein using default parameters. In certain embodiments,variants may differ from a reference sequence by about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43,44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 (including all integersin between) or more bases. In certain embodiments, such as when thepolynucleotide variant encodes a HRS polypeptide having a non-canonicalactivity, the desired activity of the encoded HRS polypeptide is notsubstantially diminished relative to the unmodified polypeptide. Theeffect on the activity of the encoded polypeptide may generally beassessed as described herein. In some embodiments, the variants canalter the aggregation state of the HRS polypeptides, for example toprovide for HRS polypeptides that exist in different embodimentsprimarily as a monomer, dimer or multimer.

Certain embodiments include polynucleotides that hybridize to areference HRS polynucleotide sequence, (such as for example, SEQ IDNOS:111-127, 182-184, 192-198; see also the Examples) or to theircomplements, under stringency conditions described below. As usedherein, the term “hybridizes under low stringency, medium stringency,high stringency, or very high stringency conditions” describesconditions for hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel et al., (1998, supra),Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described inthat reference and either can be used.

Reference herein to low stringency conditions include and encompass fromat least about 1% v/v to at least about 15% v/v formamide and from atleast about 1 M to at least about 2 M salt for hybridization at 42° C.,and at least about 1 M to at least about 2 M salt for washing at 42° C.Low stringency conditions also may include 1% Bovine Serum Albumin(BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS for washing at room temperature. One embodiment of lowstringency conditions includes hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions).

Medium stringency conditions include and encompass from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9 M salt for hybridization at 42° C., and at leastabout 0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringencyconditions include and encompass from at least about 31% v/v to at leastabout 50% v/v formamide and from about 0.01 M to about 0.15 M salt forhybridization at 42° C., and about 0.01 M to about 0.02 M salt forwashing at 55° C.

High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 MNaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC,0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS forwashing at a temperature in excess of 65° C. One embodiment of highstringency conditions includes hybridizing in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Oneembodiment of very high stringency conditions includes hybridizing in0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesin 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104. While stringent washes are typically carried out at temperaturesfrom about 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6(log₁₀+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)—15° C. for high stringency, or T_(m)—30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.

Production of HRS Polypeptides and HRS-Fc Conjugates

HRS-Fc conjugate polypeptides may be prepared by any suitable procedureknown to those of skill in the art for example, by using standardsolid-phase peptide synthesis (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)), or by recombinant technology using a geneticallymodified host. Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the desiredmolecule.

HRS-Fc conjugates can also be produced by expressing a DNA or RNAsequence encoding the HRS polypeptide or HRS-Fc conjugates in questionin a suitable host cell by well-known techniques. The polynucleotidesequence coding for the HRS-Fc conjugate or HRS polypeptide may beprepared synthetically by established standard methods, e.g., thephosphoamidite method described by Beaucage et al., Tetrahedron Letters22:1859-1869, 1981; or the method described by Matthes et al., EMBOJournal 3:801-805, 1984. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, duplexed and ligated to form the synthetic DNA construct.Alternatively the DNA or RNA construct can be constructed using standardrecombinant molecular biological techniques including restriction enzymemediated cloning and PCR based gene amplification. In some embodimentsfor direct mRNA mediated expression the polynucleotide may beencapsulated in a nanoparticle or liposome to enable efficient deliveryand uptake into the cell, and optionally include a modified cap or tailstructure to enhance stability and translation.

The polynucleotide sequences may also be of mixed genomic, cDNA, RNA,and that of synthetic origin. For example, a genomic or cDNA sequenceencoding a leader peptide may be joined to a genomic or cDNA sequenceencoding the HRS polypeptide or HRS-Fc conjugate, after which the DNA orRNA sequence may be modified at a site by inserting syntheticoligonucleotides encoding the desired amino acid sequence for homologousrecombination in accordance with well-known procedures or preferablygenerating the desired sequence by PCR using suitable oligonucleotides.In some embodiments a signal sequence can be included before the codingsequence. This sequence encodes a signal peptide N-terminal to thecoding sequence which communicates to the host cell to direct thepolypeptide to the cell surface or secrete the polypeptide into themedia. Typically the signal peptide is clipped off by the host cellbefore the protein leaves the cell. Signal peptides can be found invariety of proteins in prokaryotes and eukaryotes.

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including mammalian cell and more specifically human cellsystems transformed with viral, plasmid, episomal or integratingexpression vectors.

The “control elements” or “regulatory sequences” present in anexpression vector are non-translated regions of the vector—enhancers,promoters, 5′ and 3′ untranslated regions—which interact with hostcellular proteins to carry out transcription and translation. Suchelements may vary in their strength and specificity. Depending on thevector system and host utilized, any number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, may be used. For example, when cloning in bacterial systems,inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPTphagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL,Gaithersburg, Md.) and the like may be used. In mammalian cell systems,promoters from mammalian genes or from mammalian viruses are generallypreferred. If it is necessary to generate a cell line that containsmultiple copies of the sequence encoding a polypeptide, vectors based onSV40 or EBV may be advantageously used with an appropriate selectablemarker.

Certain embodiments may employ E. cob-based expression systems (see,e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146,2008). These and related embodiments may rely partially or totally onligation-independent cloning (LIC) to produce a suitable expressionvector. In specific embodiments, protein expression may be controlled bya T7 RNA polymerase (e.g., pET vector series), or modified pET vectorswith alternate promoters, including for example the TAC promoter. Theseand related embodiments may utilize the expression host strainBL21(DE3), a λDE3 lysogen of BL21 that supports T7-mediated expressionand is deficient in lon and ompT proteases for improved target proteinstability. Also included are expression host strains carrying plasmidsencoding tRNAs rarely used in E. coli, such as ROSETTA™ (DE3) andRosetta 2 (DE3) strains. In some embodiments other E. coli strains maybe utilized, including other E. coli K-12 strains such as W3110 (F⁻lambda⁻ IN(rrnD-rrnE)1 rph-1), and UT5600 (F, araC14, leuB6(Am),secA206(aziR), lacY1, proC14, tsx67, Δ(ompTfepC)266, entA403,glnX44(AS), λ⁻, trpE38, rfbCl, rpsL109(strR), xylA5, mtl-1, thiE1),which can result in reduced levels of post-translational modificationsduring fermentation. Cell lysis and sample handling may also be improvedusing reagents sold under the trademarks BENZONASE® nuclease andBUGBUSTER® Protein Extraction Reagent. For cell culture, auto-inducingmedia can improve the efficiency of many expression systems, includinghigh-throughput expression systems. Media of this type (e.g., OVERNIGHTEXPRESS™ Autoinduction System) gradually elicit protein expressionthrough metabolic shift without the addition of artificial inducingagents such as IPTG.

Particular embodiments employ hexahistidine tags (such as those soldunder the trademark HIS•TAG® fusions), followed by immobilized metalaffinity chromatography (IMAC) purification, or related techniques. Incertain aspects, however, clinical grade proteins can be isolated fromE. coli inclusion bodies, without or without the use of affinity tags(see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As afurther example, certain embodiments may employ a cold-shock induced E.coli high-yield production system, because over-expression of proteinsin Escherichia coli at low temperature improves their solubility andstability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882,2004).

Also included are high-density bacterial fermentation systems. Forexample, high cell density cultivation of Ralstonia eutropha allowsprotein production at cell densities of over 150 g/L, and the expressionof recombinant proteins at titers exceeding 10 g/L. In the yeastSaccharomyces cerevisiae, a number of vectors containing constitutive orinducible promoters such as alpha factor, alcohol oxidase, and PGH maybe used. For reviews, see Ausubel et al. (supra) and Grant et al.,Methods Enzymol. 153:516-544, 1987. Also included are Pichia pandorisexpression systems (see, e.g., Li et al., Nature Biotechnology. 24,210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Certainembodiments include yeast systems that are engineered to selectivelyglycosylate proteins, including yeast that have humanizedN-glycosylation pathways, among others (see, e.g., Hamilton et al.,Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol.3:119-28, 2005; and Gerngross et al., Nature-Biotechnology.22:1409-1414, 2004; U.S. Pat. Nos. 7,629,163; 7,326,681; and 7,029,872).Merely by way of example, recombinant yeast cultures can be grown inFernbach Flasks or 15 L, 50 L, 100 L, and 200 L fermentors, amongothers.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311, 1987).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680,1984; Broglie et al., Science. 224:838-843, 1984; and Winter et al.,Results Probl. Cell Differ. 17:85-105, 1991). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196, 1992).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia cells. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusiacells in which the polypeptide of interest may be expressed (Engelhardet al., PNAS USA. 91:3224-3227, 1994). Also included are baculovirusexpression systems, including those that utilize SF9, SF21, and T. nicells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci.Chapter 5:Unit5.4, 2001). Insect systems can provide post-translationmodifications that are similar to mammalian systems.

In mammalian host cells, a number of expression systems are well knownin the art and commercially available. Exemplary mammalian vectorsystems include for example, pCEP4, pREP4, and pREP7 from Invitrogen,the PerC6 system from Crucell, and Lentiviral based systems such as pLP1from Invitrogen, and others. For example, in cases where an adenovirusis used as an expression vector, sequences encoding a polypeptide ofinterest may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, PNAS USA.81:3655-3659, 1984). In addition, transcription enhancers, such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Examples of useful mammalian host cell lines include monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidneyline (293 or 293 cells sub-cloned for growth in suspension culture,Graham et al., J. Gen Vivol. 36:59, 1977); baby hamster kidney cells(BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA.77:4216, 1980); and myeloma cell lines such as NSO and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.Certain preferred mammalian cell expression systems include CHO andHEK293-cell based expression systems. Mammalian expression systems canutilize attached cell lines, for example, in T-flasks, roller bottles,or cell factories, or suspension cultures, for example, in 1 L and 5 Lspinners, 5L, 14L, 40L, 100 L and 200L stir tank bioreactors, or 20/50 Land 100/200 L WAVE bioreactors, among others known in the art.

Also included are methods of cell-free protein expression. These andrelated embodiments typically utilize purified RNA polymerase,ribosomes, tRNA, and ribonucleotides. Such reagents can be produced, forexample, by extraction from cells or from a cell-based expressionsystem.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, post-translationalmodifications such as acetylation, carboxylation, glycosylation,phosphorylation, lipidation, and acylation, or the insertion ofnon-naturally occurring amino acids (see generally U.S. Pat. Nos.7,939,496; 7,816,320; 7,947,473; 7,883,866; 7,838,265; 7,829,310;7,820,766; 7,820,766; 7,7737,226, 7,736,872; 7,638,299; 7,632,924; and7,230,068). In some embodiments, such non-naturally occurring aminoacids may be inserted at position Cys130. Post-translational processingwhich cleaves a “prepro” form of the protein may also be used tofacilitate correct insertion, folding and/or function. Different hostcells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition tobacterial cells, which have or even lack specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

The HRS polypeptides or HRS-Fc conjugates produced by a recombinant cellcan be purified and characterized according to a variety of techniquesknown in the art. Exemplary systems for performing protein purificationand analyzing protein purity include fast protein liquid chromatography(FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquidchromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplarychemistries for purification include ion exchange chromatography (e.g.,Q, S), size exclusion chromatography, salt gradients, affinitypurification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G),gel filtration, reverse-phase, ceramic HYPERD® ion exchangechromatography, and hydrophobic interaction columns (HIC), among othersknown in the art. Several exemplary methods are also disclosed in theExamples sections.

HRS-Fc Conjugates

As noted above, embodiments of the present invention relate to HRS-Fcconjugates, which comprise at least one Fc region that is covalentlyattached to one or more HRS polypeptides. Examples of HRS-Fc conjugatesinclude fusion proteins and various forms of chemically cross-linkedproteins. A wide variety of Fc region sequences may be employed in theHRS-Fc conjugates of the present invention, including wild-typesequences from any number of species, as well as variants, fragments,hybrids, and chemically modified forms thereof. The HRS-Fc polypeptidesmay also (optionally) comprise one or more linkers, which typicallyseparate the Fc region(s) from the HRS polypeptide(s), including peptidelinkers and chemical linkers, as described herein and known in the art.It will be appreciated that in any of these HRS-Fc conjugates the nativeN or C terminal amino acid of the HRS polypeptides, or native N orC-amino acid in the Fc domain, may be deleted and/or replaced with nonnative amino acid(s), for example, to facilitate expression and orcloning or to serve as a linker sequence between the two proteins.

HRS-Fc conjugate polypeptides can provide a variety of advantagesrelative to un-conjugated or unmodified HRS polypeptides, e.g.,corresponding HRS polypeptides of the same or similar sequence having noFc region(s) attached thereto. Merely by way of illustration, thecovalent attachment of one or more Fc regions can alter (e.g., increase,decrease) the HRS polypeptide's solubility, half-life (e.g., in serum,in a selected tissue, in a test tube under storage conditions, forexample, at room temperature or under refrigeration), dimerization ormultimerization properties, biological activity or activities, forinstance, by providing Fc-region-associated effector functions (e.g.,activation of the classical complement cascade, interaction with immuneeffector cells via the Fc receptor (FcR), compartmentalization ofimmunoglobulins), cellular uptake, intracellular transport, tissuedistribution, and/or bioavailability, relative to an unmodified HRSpolypeptide having the same or similar sequence. In certain aspects, Fcregions can confer effector functions relating to complement-dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), and/or antibody-dependent cell-mediated phagocytocis (ADCP),which are believed to play a role in clearing specific target cells suchas tumor cells and infected cells.

Certain embodiments employ HRS-Fc fusion proteins. “Fusion proteins” aredefined elsewhere herein and well known in the art, as are methods ofmaking fusion proteins (see, e.g., U.S. Pat. Nos. 5,116,964; 5,428,130;5,455,165; 5,514,582; 6,406,697; 6,291,212; and 6,300,099 for generaldisclosure and methods related to Fc fusion proteins). In a HRS-Fcfusion protein, the Fc region can be fused to the N-terminus of the HRSpolypeptide, the C-terminus, or both. In some embodiments, one or moreFc regions can be fused internally relative to HRS sequences, forinstance, by placing an Fc region between a first HRS sequence (e.g.,domain) and a second HRS sequence (e.g., domain), where the first HRSsequence is fused to the N-terminus of the Fc region and the second HRSsequence is fused to the C-terminus of the Fc region. In specificembodiments, the first and second HRS sequences are identical. In otherembodiments, the first and second HRS sequences are different (e.g.,they include different functional domains of the HRS polypeptide).Certain HRS-Fc fusion proteins can also include additional heterologousprotein sequences, that is, non-Fc region and non-HRS polypeptidesequences.

The term “HRS-Fc” can indicate, but does not necessarily indicate, theN-terminal or C-terminal attachment of the Fc region to the HRSpolypeptide. For instance, in certain instances the term “Fc-HRS”indicates fusion of the Fc region to the N-terminus of the HRSpolypeptide, and the term “HRS-Fc” indicates fusion of the Fc region tothe C-terminus of the HRS polypeptide. However, either term can be usedmore generally to refer to any fusion protein or conjugate of an Fcregion and a HRS polypeptide.

In some embodiments the HRS-Fc fusion proteins may comprise tandemlyrepeated copies of the HRS polypeptide coupled to a single Fc domain,optionally separated by linker peptides. Exemplary tandemly repeatedHRS-Fc fusion proteins are provided in Table D10. The preparation andsequences for specific tandemly repeated HRS-Fc conjugates areillustrated in the Examples.

TABLE D10 Exemplary Tandem HRS-Fc conjugates HRSpolypeptide-L-HRS-polypeptide-L-Fc HRS-polypeptide-L-HRS-polypeptide-L-HRS-polypeptide-L-FcHRS-polypeptide-L-HRS-polypeptide-L-HRS-polypeptide-L-HRS- polypeptide-L-Fc Fc-L-HRS-polypeptide -L-HRS-polypeptide Fc-L-HRS-polypeptide-L-HRS-L-HRS-polypeptide Fc-L-HRS-polypeptide-L-HRS-L-HRS-L-HRS-polypeptide Where: “Fc” is an Fc domain as describedherein. “HRS-polypeptide” is any of the truncated HRS polypeptidesdescribed in Table D5. “L” is an optional peptide linker.

Certain embodiments relate to HRS-Fc conjugates, where, for instance,one or more Fc regions are chemically conjugated or cross-linked to theHRS polypeptide(s). In these and related aspects, the Fc region can beconjugated to the HRS polypeptide at the N-terminal region (e.g., withinthe first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or so amino acids),the internal region (between the N-terminal and C-terminal regions),and/or the C-terminal region (e.g., within the last 10, 20, 30, 40, 50,60, 70, 80, 90, 100 or so amino acids). Polypeptides can be conjugatedor cross-linked to other polypeptides according to a variety of routinetechniques in the art. For instance, certain techniques employ thecarboxyl-reactive carbodiimide crosslinker EDC (or EDAC), whichcovalently attaches via D, E, and C-terminal carboxyl groups. Othertechniques employ activated EDC, which covalently attaches via K andN-terminal amino groups). Still other techniques employm-maleimidobenzoyl-N-hydoxysuccinimide ester (MBS) or Sulfo-MBS, whichcovalently attach via the thiol group of a cysteine residue (see alsoU.S. Application No. 2007/0092940 for cysteine engineered Ig regionsthat can be used for thiol conjugation). Such cross-linked proteins canalso comprise linkers, including cleavable or otherwise releasablelinkers (e.g., enzymatically cleavable linkers, hydrolysable linkers),and non-cleavable linkers (i.e., physiologically-stable linkers).Certain embodiments may employ non-peptide polymers (e.g., PEG polymers;HRS-N-PEG-N-Fc conjugate) as a cross-linker between the Fc region(s) andthe HRS polypeptide(s), as described, for example, in U.S. ApplicationNo. 2006/0269553. See also US Application No. 2007/0269369 for exemplarydescriptions of Fc region conjugation sites.

In certain embodiments, discussed in greater detail below, variant orotherwise modified Fc regions can be employed, including those havingaltered properties or biological activities relative to wild-type Fcregion(s). Examples of modified Fc regions include those having mutatedsequences, for instance, by substitution, insertion, deletion, ortruncation of one or more amino acids relative to a wild-type sequence,hybrid Fc polypeptides composed of domains from different immunoglobulinclasses/subclasses, Fc polypeptides having alteredglycosylation/sialylation patterns, and Fc polypeptides that aremodified or derivatized, for example, by biotinylation (see, e.g., USApplication No. 2010/0209424), phosphorylation, sulfation, etc., or anycombination of the foregoing. Such modifications can be employed toalter (e.g., increase, decrease) the binding properties of the Fc regionto one or more particular FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc,FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g.,stability or half-life, bioavailability, tissue distribution, volume ofdistribution, concentration, elimination rate constant, eliminationrate, area under the curve (AUC), clearance, C., t_(max), C_(min),fluctuation), its immunogenicity, its complement fixation or activation,and/or the CDC/ADCC/ADCP-related activities of the Fc region, amongother properties described herein, relative to a corresponding wild-typeFc sequence.

The “Fc region” of a HRS-Fc conjugate provided herein is usually derivedfrom the heavy chain of an immunoglobulin (Ig) molecule. A typical Igmolecule is composed of two heavy chains and two light chains. The heavychains can be divided into at least three functional regions: the Fdregion, the Fc region (fragment crystallizable region), and the hingeregion (see FIG. 1), the latter being found only in IgG, IgA, and IgDimmunoglobulins. The Fd region comprises the variable (V_(H)) andconstant (CH₁) domains of the heavy chains, and together with thevariable (V_(L)) and constant (C_(L)) domains of the light chains formsthe antigen-binding fragment or Fab region.

The Fc region of IgG, IgA, and IgD immunoglobulins comprises the heavychain constant domains 2 and 3, designated respectively as CH₂ and CH₃regions; and the Fc region of IgE and IgM immunoglobulins comprises theheavy chain constant domains 2, 3, and 4, designated respectively asCH₂, CH₃, and CH₄ regions. The Fc region is mainly responsible for theimmunoglobulin effector functions, which include, for example,complement fixation and binding to cognate Fc receptors of effectorcells.

The hinge region (found in IgG, IgA, and IgD) acts as a flexible spacerthat allows the Fab portion to move freely in space relative to the Fcregion. In contrast to the constant regions, the hinge regions arestructurally diverse, varying in both sequence and length amongimmunoglobulin classes and subclasses. The hinge region may also containone or more glycosylation site(s), which include a number ofstructurally distinct types of sites for carbohydrate attachment. Forexample, IgA1 contains five glycosylation sites within a 17 amino acidsegment of the hinge region, conferring significant resistance of thehinge region polypeptide to intestinal proteases. Residues in the hingeproximal region of the CH₂ domain can also influence the specificity ofthe interaction between an immunoglobulin and its respective Fcreceptor(s) (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186,1993).

The term “Fc region” or “Fc fragment” or “Fc” as used herein, thusrefers to a protein that contains one or more of a CH₂ region, a CH₃region, and/or a CH₄ region from one or more selected immunoglobulin(s),including fragments and variants and combinations thereof. An “Fcregion” may also include one or more hinge region(s) of the heavy chainconstant region of an immunoglobulin. In certain embodiments, the Fcregion does not contain one or more of the CH₁, C_(L), V_(L), and/orV_(H) regions of an immunoglobulin.

The Fc region can be derived from the CH₂ region, CH₃ region, CH₄region, and/or hinge region(s) of any one or more immunoglobulinclasses, including but not limited to IgA, IgD, IgE, IgG, IgM, includingsubclasses and combinations thereof. In some embodiments, the Fc regionis derived from an IgA immunoglobulin, including subclasses IgA1 and/orIgA2. In certain embodiments, the Fc region is derived from an IgDimmunoglobulin. In particular embodiments, the Fc region is derived froman IgE immunoglobulin. In some embodiments, the Fc region is derivedfrom an IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3,and/or IgG4. In certain embodiments, the Fc region is derived from anIgM immunoglobulin. FIG. 2 shows an alignment of Fc regions from humanIgA1 (SEQ ID NO:156), IgA2 (SEQ ID NO:157), IgM (SEQ ID NO:158), IgG1(SEQ ID NO:159), IgG2 (SEQ ID NO:160), IgG3 (SEQ ID NO:161), IgG4(SEQ IDNO:162), and IgE (SEQ ID NO:163).

Certain Fc regions demonstrate specific binding for one or moreFc-receptors (FcRs). Examples of classes of Fc receptors include Fcγreceptors (FcγR), Fca receptors (FcαR), Fcε receptors (FcεR), and theneonatal Fc receptor (FcRn). For instance, certain Fc regions haveincreased binding to (or affinity for) one or more FcγRs, relative toFcαRs, FcγRs, and/or FcRn. In some embodiments, Fc regions haveincreased binding to FcαRs, relative to one or more FcγRs, FcγRs, and/orFcRn. In other embodiments, Fc regions have increased binding to FcγRs(e.g., FcαRI), relative to one or more FcγRs, FcαRs, and/or FcRn. Inparticular embodiments, Fc regions have increased binding to FcRn,relative to one or more FcγRs, FcαRs, and/or FcγRs. In certainembodiments, the binding (or affinity) of an Fc region to one or moreselected FcR(s) is increased relative to its binding to (or affinityfor) one or more different FcR(s), typically by about 1.5×, 2×, 2.5×,3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×,50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×,900×, 1000× or more (including all integers in between).

Examples of FcγRs include FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa,and FcγRIIIb. FcγRI (CD64) is expressed on macrophages and dendriticcells and plays a role in phagocytosis, respiratory burst, cytokinestimulation, and dendritic cell endocytic transport. Expression of FcγRIis upregulated by both GM-CSF and γ-interferon (γ-IFN) and downregulatedby interleukin-4 (IL-4). FcγRIIa is expressed on polymorphonuclearleukocytes (PMN), macrophages, dendritic cells, and mast cells. FcγRIIaplays a role in phagocytosis, respiratory burst, and cytokinestimulation. Expression of FcγRIIa is upregulated by GM-CSF and γ-IFN,and decreased by IL-4. FcγIIb is expressed on B cells, PMN, macrophages,and mast cells. FcγIIb inhibits immunoreceptor tyrosine-based activationmotif (ITAM) mediated responses, and is thus an inhibitory receptor.Expression of FcγRIIc is upregulated by intravenous immunoglobulin(IVIG) and IL-4 and decreased by γ-IFN. FcγRIIc is expressed on NKcells. FcγRIIIa is expressed on natural killer (NK) cells, macrophages,mast cells, and platelets. This receptor participates in phagocytosis,respiratory burst, cytokine stimulation, platelet aggregation anddegranulation, and NK-mediated ADCC. Expression of FcγRIII isupregulated by C5a, TGF-β, and γ-IFN and downregulated by IL-4. Fc γRIIIb is a GPI-linked receptor expressed on PMN.

Certain Fc regions have increased binding to FcγRI, relative to FcγRIIa,FcγRIIb, FcγRIIc, FcγRIIIa, and/or FcγRIIIb. Some embodiments haveincreased binding to FcγRIIa, relative to FcγRI, FcγRIIb, FcγRIIc,FcγRIIIa, and/or FcγRIIIb. Particular Fc regions have increased bindingto FcγRIIb, relative to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and/orFcγRIIIb. Certain Fc regions have increased binding to FcγRIIc, relativeto FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and/or FcγRIIIb. Some Fc regionshave increased binding to FcγRIIIa, relative to FcγRI, FcγRIIa, FcγRIIb,FcγRIIc, and/or FcγRIIIb. Specific Fc regions have increased binding toFcγRIIIb, relative to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and/or FcγRIIIa.

FcαRs include FcαRI (CD89). FcαRI is found on the surface ofneutrophils, eosinophils, monocytes, certain macrophages (e.g., Kupffercells), and certain dendritic cells. FcαRI is composed of twoextracellular Ig-like domains, is a member of both the immunoglobulinsuperfamily and the multi-chain immune recognition receptor (MIRR)family, and signals by associating with two FcRγ signaling chains.

FcαRs include FcεRI and FcεRII. The high-affinity receptor FcεRI is amember of the immunoglobulin superfamily, is expressed on epidermalLangerhans cells, eosinophils, mast cells and basophils, and plays amajor role in controlling allergic responses. FcεRI is also expressed onantigen-presenting cells, and regulates the production pro-inflammatorycytokines. The low-affinity receptor FcεRII (CD23) is a C-type lectinthat can function as a membrane-bound or soluble receptor. FcεRIIregulates B cell growth and differentiation, and blocks IgE-binding ofeosinophils, monocytes, and basophils. Certain Fc regions have increasedbinding to FcεRI, relative to FcεRII. Other Fc regions have increasedbinding to FcεRII, relative to FcεRI.

Table F1 below summarizes the characteristics of certain FcRs.

TABLE F1 Exemplary Fc-Receptors Primary Exemplary Effects AntibodyFollowing Binding Receptor Ligand Ligand Affinity Cell Distribution toFc Ligand FcγRI IgG1 and High (Kd~10⁻⁹ M) Macrophages Phagocytosis(CD64) IgG3 Neutrophils Cell activation Eosinophils Activation ofrespiratory burst Dendritic cells Induction of microbe killing FcγRIIaIgG Low (Kd > 10⁻⁷ M) Macrophages Phagocytosis (CD32) NeutrophilsDegranulation (eosinophils) Eosinophils Platelets Langerhans cellsFcγRIIb1 IgG Low (Kd > 10⁻⁷ M) B Cells No phagocytosis (CD32) Mast cellsInhibition of cell activity FcγRIIb2 IgG Low (Kd > 10⁻⁷ M) MacrophagesPhagocytosis (CD32) Neutrophils Inhibition of cell activity EosinophilsFcγRIIIa IgG Low (Kd > 10⁻⁶ M) NK cells Induction of antibody-dependentcell- (CD16a) Macrophages mediated cytotoxicity (ADCC) (certain tissues)Induction of cytokine release by macrophages FcγRIIIb IgG Low (Kd > 10⁻⁶M) Eosinophils Induction of microbe killing (CD16b) MacrophagesNeutrophils Mast cells Follicular dendritic cells FcεRI IgE High(Kd~10⁻¹⁰ M) Mast cells Degranulation Eosinophils Basophils Langerhanscells FcεRII IgE Low (Kd > 10⁻⁷ M) B cells Possible adhesion molecule(CD23) Eosinophils Langerhans cells FcαRI IgA Low (Kd > 10⁻⁶ M)Monocytes Phagocytosis (CD89) Macrophages Induction of microbe killingNeutrophils Eosinophils Fcα/μR IgA and High for IgM, B cells EndocytosisIgM Moderate for IgA Mesangial cells Induction of microbe killingMacrophages FcRn IgG Monocytes Transfers IgG from a mother to fetusMacrophages through the placenta Dendrite cells Transfers IgG from amother to infant Epithelial cells in milk Endothelial cells Protects IgGfrom degradation Hepatocytes

Fc regions can be derived from the immunoglobulin molecules of anyanimal, including vertebrates such as mammals such cows, goats, swine,dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates,and humans. The amino acid sequences of CH₂, CH₃, CH₄, and hinge regionsfrom exemplary, wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3,IgG4, and IgM immunoglobulins are shown below (SEQ ID NOS:128-154).

SEQ ID NO: 128 is the amino acid sequence of a human IgA1 hinge region(VPSTPPTPSPSTPPTPSPS).SEQ ID NO: 129 is the amino acid sequence of a human IgA1 CH2 region(CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS).SEQ ID NO: 130 is the amino acid sequence of a human IgA1 CH3 region(GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVD GTCY).SEQ ID NO: 131 is the amino acid sequence of a human IgA2 hinge region (VPPPPP).SEQ ID NO: 132 is the amino acid sequence of a human IgA2 CH2 region(CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKS).SEQ ID NO: 133 is the amino acid sequence of a human IgA2 CH3 region(GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVD GTCY).SEQ ID NO: 134 is the amino acid sequence of a human IgD hinge region(ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP).SEQ ID NO: 135 is the amino acid sequence of a human IgD CH2 region(ECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREP).SEQ ID NO: 136 is the amino acid sequence of a human IgD CH3 region(AAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK).SEQ ID NO: 137 is the amino acid sequence of a human IgE CH2 region(VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCA).SEQ ID NO: 138 is the amino acid sequence of a human IgE CH3 region(DSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS).SEQ ID NO: 139 is the amino acid sequence of a human IgE CH4 region(GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVIRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK).SEQ ID NO: 140 is the amino acid sequence of a human IgG1 hinge region(EPKSCDKTHTCPPCP).SEQ ID NO: 341 is the amino acid sequence of a modified human IgG1 hinge region derivedsequence (SDKTHTCPPCP).SEQ ID NO: 141 is the amino acid sequence of a human IgG1 CH2 region(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK).SEQ ID NO: 142 is the amino acid sequence of a human IgG1 CH3 region(GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).SEQ ID NO: 342 is the amino acid sequence of a human IgG1 heavy chain sequence(MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). It will be appreciated that the Met residuein this human IgG1 heavy chain sequence can be deleted, for instance, upon N-terminalfusion to a HRS polypeptide (see SEQ ID NO: 340).SEQ ID NO: 143 is the amino acid sequence of a human IgG2 hinge region (ERKCCVECPPCP).SEQ ID NO: 144 is the amino acid sequence of a human IgG2 CH2 region(APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK).SEQ ID NO: 145 is the amino acid sequence of a human IgG2 CH3 region(GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).SEQ ID NO: 146 is the amino acid sequence of a human IgG3 hinge region(ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP).SEQ ID NO: 147 is the amino acid sequence of a human IgG3 CH2 region(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK).SEQ ID NO: 148 is the amino acid sequence of a human IgG3 CH3 region(GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK).SEQ ID NO: 149 is the amino acid sequence of a human IgG4 hinge region (ESKYGPPCPSCP).SEQ ID NO: 150 is the amino acid sequence of a human IgG4 CH2 region(APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK).SEQ ID NO: 151 is the amino acid sequence of a human IgG4 CH3 region(GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).SEQ ID NO: 152 is the amino acid sequence of a human IgM CH2 region(VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP).SEQ ID NO: 153 is the amino acid sequence of a human IgM CH3 region(DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK).SEQ ID NO: 154 is the amino acid sequence of a human IgM CH4 region(GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAG TCY).

A HRS-Fc conjugate of the present invention can thus comprise, consistof, or consist essentially of one or more of the human Fc region aminoacid sequences of SEQ ID NOS:128-163 or 339-342, including variants,fragments, homologs, orthologs, paralogs, and combinations thereof.Certain illustrative embodiments comprise an Fc region that ranges insize from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400,50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200,100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or200-400 amino acids in length, and optionally comprises, consists of, orconsists essentially of any one or more of SEQ ID NOS:128-154 or341-342. Certain embodiments comprise an Fc region of up to about 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, whichoptionally comprises, consists of, or consists essentially of any one ormore of SEQ ID NOS: 128-154 or 339-342.

Certain Fc regions comprise, consist of, or consist essentially of humanIgA1 sequences set forth in SEQ ID NOS:128-130 or 156, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:128 and 129 and 130, SEQ ID NOS:128 and 129; SEQ IDNOS:128 and 130; SEQ ID NOS:129 and 130), and variants and fragmentsthereof. Certain Fc regions comprise, consist of, or consist essentiallyof human the IgA1 sequence set forth in SEQ ID NOS:128. Certain Fcregions comprise, consist of, or consist essentially of the human IgA1sequence set forth in SEQ ID NOS:129. Certain Fc regions comprise,consist of, or consist essentially of the human IgA1 sequence set forthin SEQ ID NOS:130.

Some Fc regions comprise, consist of, or consist essentially of humanIgA2 sequences set forth in SEQ ID NOS:131-133 or 157, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:131 and 132 and 133, SEQ ID NOS:131 and 132; SEQ IDNOS:131 and 133; SEQ ID NOS:132 and 133), and variants and fragmentsthereof. Certain Fc regions comprise, consist of, or consist essentiallyof human the IgA2 sequence set forth in SEQ ID NOS:131. Certain Fcregions comprise, consist of, or consist essentially of the human IgA2sequence set forth in SEQ ID NOS:132. Certain Fc regions comprise,consist of, or consist essentially of the human IgA2 sequence set forthin SEQ ID NOS:133.

Certain Fc regions comprise, consist of, or consist essentially of humanIgD sequences set forth in SEQ ID NOS:134-136, in any order reading fromN-terminus to C-terminus, including combinations thereof (e.g., SEQ IDNOS:134 and 135 and 136, SEQ ID NOS:134 and 135; SEQ ID NOS:134 and 136;SEQ ID NOS:135 and 136), and variants and fragments of these sequencesand combinations. Certain Fc regions comprise, consist of, or consistessentially of human the IgD sequence set forth in SEQ ID NOS:134.Certain Fc regions comprise, consist of, or consist essentially of thehuman IgD sequence set forth in SEQ ID NOS:135. Certain Fc regionscomprise, consist of, or consist essentially of the human IgD sequenceset forth in SEQ ID NOS:136.

Certain Fc regions comprise, consist of, or consist essentially of humanIgE sequences set forth in SEQ ID NOS:137-139 or 163, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:137 and 138 and 139, SEQ ID NOS:137 and 138; SEQ IDNOS:137 and 139; SEQ ID NOS:138 and 139), and variants and fragments ofthese sequences and combinations. Certain Fc regions comprise, consistof, or consist essentially of human the IgE sequence set forth in SEQ IDNOS:137. Certain Fc regions comprise, consist of, or consist essentiallyof the human IgE sequence set forth in SEQ ID NOS:138. Certain Fcregions comprise, consist of, or consist essentially of the human IgEsequence set forth in SEQ ID NOS:139.

Certain Fc regions comprise, consist of, or consist essentially of humanIgG1 sequences set forth in SEQ ID NOS:140-142 or 159 or 339-342, in anyorder reading from N-terminus to C-terminus, including combinationsthereof (e.g., SEQ ID NOS:140 and 141 and 142, SEQ ID NOS:140 and 141;SEQ ID NOS:140 and 142; SEQ ID NOS:141 and 142), and variants andfragments of these sequences and combinations. Certain Fc regionscomprise, consist of, or consist essentially of human the IgG1 sequenceset forth in SEQ ID NOS:140. Certain Fc regions comprise, consist of, orconsist essentially of the human IgG1 sequence set forth in SEQ IDNOS:141. Certain Fc regions comprise, consist of, or consist essentiallyof the human IgG1 sequence set forth in SEQ ID NOS:142. Certain Fcregions comprise, consist of, or consist essentially of the human IgG1sequence set forth in SEQ ID NOS:339. Certain Fc regions comprise,consist of, or consist essentially of the human IgG1 sequence set forthin SEQ ID NOS:340. Certain Fc regions comprise, consist of, or consistessentially of the human IgG1 sequence set forth in SEQ ID NOS:341.Certain Fc regions comprise, consist of, or consist essentially of thehuman IgG1 sequence set forth in SEQ ID NOS:342.

Certain Fc regions comprise, consist of, or consist essentially of humanIgG2 sequences set forth in SEQ ID NOS:143-145 or 160, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:143 and 144 and 145, SEQ ID NOS:143 and 144; SEQ IDNOS:143 and 145; SEQ ID NOS:144 and 145), and variants and fragments ofthese sequences and combinations. Certain Fc regions comprise, consistof, or consist essentially of human the IgG2 sequence set forth in SEQID NOS:143. Certain Fc regions comprise, consist of, or consistessentially of the human IgG2 sequence set forth in SEQ ID NOS:144.Certain Fc regions comprise, consist of, or consist essentially of thehuman IgG2 sequence set forth in SEQ ID NOS:145.

Certain Fc regions comprise, consist of, or consist essentially of humanIgG3 sequences set forth in SEQ ID NOS:146-148 or 161, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:146 and 147 and 148, SEQ ID NOS:146 and 147; SEQ IDNOS:146 and 148; SEQ ID NOS:147 and 148), and variants and fragments ofthese sequences and combinations. Certain Fc regions comprise, consistof, or consist essentially of human the IgG3 sequence set forth in SEQID NOS:146. Certain Fc regions comprise, consist of, or consistessentially of the human IgG3 sequence set forth in SEQ ID NOS:147.Certain Fc regions comprise, consist of, or consist essentially of thehuman IgG3 sequence set forth in SEQ ID NOS:148.

Certain Fc regions comprise, consist of, or consist essentially of humanIgG4 sequences set forth in SEQ ID NOS:149-151 or 162, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:149 and 150 and 151, SEQ ID NOS:149 and 150; SEQ IDNOS:149 and 151; SEQ ID NOS:150 and 151), and variants and fragments ofthese sequences and combinations. Certain Fc regions comprise, consistof, or consist essentially of human the IgG4 sequence set forth in SEQID NOS:149. Certain Fc regions comprise, consist of, or consistessentially of the human IgG4 sequence set forth in SEQ ID NOS:150.Certain Fc regions comprise, consist of, or consist essentially of thehuman IgG4 sequence set forth in SEQ ID NOS:151.

Certain Fc regions comprise, consist of, or consist essentially of humanIgM sequences set forth in SEQ ID NOS:152-154 or 158, in any orderreading from N-terminus to C-terminus, including combinations thereof(e.g., SEQ ID NOS:152 and 153 and 154, SEQ ID NOS:152 and 153; SEQ IDNOS:152 and 154; SEQ ID NOS:153 and 154), and variants and fragments ofthese sequences and combinations. Certain Fc regions comprise, consistof, or consist essentially of human the IgM sequence set forth in SEQ IDNOS:152. Certain Fc regions comprise, consist of, or consist essentiallyof the human IgM sequence set forth in SEQ ID NOS:153. Certain Fcregions comprise, consist of, or consist essentially of the human IgMsequence set forth in SEQ ID NOS:154.

As noted above, certain embodiments employ variants, fragments, hybrids,and/or otherwise modified forms an Fc region described herein and knownin the art (e.g., the human Ig sequences of SEQ ID NOS:128-163).

Included are variants having one or more amino acid substitutions,insertions, deletions, and/or truncations relative to a referencesequence, such as any one or more of the reference sequences set forthin SEQ ID NOS:128-163. In certain embodiments, a variant Fc regionincludes an amino acid sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ormore sequence identity or similarity or homology to any one or more ofSEQ ID NOS:128-163. Also included are Fc regions differing from one ormore of SEQ ID NOS:128-163 by the addition, deletion, insertion, orsubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150or more amino acids. In certain embodiments, the amino acid additions ordeletions occur at the C-terminal end and/or the N-terminal end of theFc reference sequence.

In particular embodiments, a variant Fc region comprises an amino acidsequence that can be optimally aligned with any one or more of SEQ IDNOS:128-163 to generate a BLAST bit scores or sequence similarity scoresof at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, or more, including all integers and ranges in between, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1.

Also included are hybrid Fc regions, for example, Fc regions thatcomprise a combination of Fc domains (e.g., hinge, CH₂, CH₃, CH₄) fromimmunoglobulins of different species, different Ig classes, and/ordifferent Ig subclasses. General examples include hybrid Fc regions thatcomprise, consist of, or consist essentially of the followingcombination of CH₂/CH₃ domains: IgA1/IgA1, IgA1/IgA2, IgA1/IgD,IgA1/IgE, IgA1/IgG1, IgA1/IgG2, IgA1/IgG3, IgA1/IgG4, IgA1/IgM,IgA2/IgA1, IgA2/IgA2, IgA2/IgD, IgA2/IgE, IgA2/IgG1, IgA2/IgG2,IgA2/IgG3, IgA2/IgG4, IgA2/IgM, IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE,IgD/IgG1, IgD/IgG2, IgD/IgG3, IgD/IgG4, IgD/IgM, IgE/IgA1, IgE/IgA2,IgE/IgD, IgE/IgE, IgE/IgG2, IgE/IgG3, IgE/IgG4, IgE/IgM, IgG1/IgA1,IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG2, IgG1/IgG3, IgG1/IgG4,IgG1/IgM, IgG2/IgA1, IgG2/IgA2, IgG2/IgD, IgG2/IgE, IgG2/IgG1,IgG2/IgG2, IgG2/IgG3, IgG2/IgG4, IgG2/IgM, IgG3/IgA1, IgG3/IgA2,IgG3/IgD, IgG3/IgE, IgG3/IgG1, IgG3/IgG2, IgG3/IgG3, IgG3/IgG4,IgG3/IgM, IgG4/IgA1, IgG4/IgA2, IgG4/IgD, IgG4/IgE, IgG4/IgG1,IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM, IgM/IgA1, IgM/IgA2, IgM/IgD,IgM/IgE, IgM/IgG1, IgM/IgG2, IgM/IgG3, IgM/IgG4, IgM/IgM (or fragmentsor variants thereof), and optionally include a hinge from one or more ofIgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and/or a CH₄ domain from IgEand/or IgM. In specific embodiments, the hinge, CH₂, CH₃, and CH₄domains are from human Ig.

Additional examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of CH₂/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG2/IgM,IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variants thereof), andoptionally include a hinge from one or more of IgA1, IgA2, IgD, IgG1,IgG2, IgG3, IgG4, and/or a CH₃ domain from one or more of IgA1, IgA2,IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specific embodiments, thehinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of CH₃/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG1/IgM,IgG2/IgM, IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variantsthereof), and optionally include a hinge from one or more of IgA1, IgA2,IgD, IgG1, IgG2, IgG3, IgG4, and/or a CH₂ domain from one or more ofIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Particular examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of hinge/CH₂domains: IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₃ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₃ domains:IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₂ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Some examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₄ domains:IgA1/IgE, IgA1/IgM, IgA2/IgE, IgA2/IgM, IgD/IgE, IgD/IgM, IgG1/IgE,IgG1/IgM, IgG2/IgE, IgG2/IgM, IgG3/IgE, IgG3/IgM, IgG4/IgE, IgG4/IgM (orfragments or variants thereof), and optionally include a CH₂ domain fromone or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM,and/or a CH₃ domain from one or more of IgA1, IgA2, IgD, IgE, IgG1,IgG2, IgG3, IgG4, or IgM.

Specific examples of hybrid Fc regions can be found, for example, in WO2008/147143, which are derived from combinations of IgG subclasses orcombinations of human IgD and IgG.

Also included are derivatized or otherwise modified Fc regions. Incertain aspects, the Fc region may be modified by phosphorylation,sulfation, acrylation, glycosylation, methylation, farnesylation,acetylation, amidation, and the like, for instance, relative to awild-type or naturally-occurring Fc region. In certain embodiments, theFc region may comprise wild-type or native glycosylation patterns, oralternatively, it may comprise increased glycosylation relative to anative form, decreased glycosylation relative to a native form, or itmay be entirely deglycosylated. As one example of a modified Fcglycoform, decreased glycosylation of an Fc region reduces binding tothe C1q region of the first complement component C1, a decrease inADCC-related activity, and/or a decrease in CDC-related activity.Certain embodiments thus employ a deglycosylated or aglycosylated Fcregion. See, e.g., WO 2005/047337 for the production of exemplaryaglycosylated Fc regions. Another example of an Fc region glycoform canbe generated by substituting the Q295 position with a cysteine residue(see, e.g., U.S. Application No. 2010/0080794), according to the Kabatet al. numbering system. Certain embodiments may include Fc regionswhere about 80-100% of the glycoprotein in Fc region comprises a maturecore carbohydrate structure that lacks fructose (see, e.g., U.S.Application No. 2010/0255013). Some embodiments may include Fc regionsthat are optimized by substitution or deletion to reduce the level offucosylation, for instance, to increase affinity for FcγRI, FcγRIa, orFcγRIIIa, and/or to improve phagocytosis by FcγRIIa-expressing cells(see U.S. Application Nos. 2010/0249382 and 2007/0148170).

As another example of a modified Fc glycoform, an Fc region may compriseoligomannose-type N-glycans, and optionally have one or more of thefollowing: increased ADCC activity, increased binding affinity forFcγRIIIA (and certain other FcRs), similar or increased bindingspecificity for the target of the HRS polypeptide, similar or higherbinding affinity for the target of the HRS polypeptide, and/or similaror lower binding affinity for mannose receptor, relative to acorresponding Fc region or HRS-Fc conjugate that contains complex-typeN-glycans (see, e.g., U.S. Application No. 2007/0092521 and U.S. Pat.No. 7,700,321). As another example, enhanced affinity of Fc regions forFcγRs has been achieved using engineered glycoforms generated byexpression of antibodies in engineered or variant cell lines (see, e.g.,Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al.,Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem.277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem.278:3466-3473, 2003; and U.S. Application No. 2007/0111281). Certain Fcregion glycoforms comprise an increased proportion of N-glycoside bondtype complex sugar chains, which do not have the 1-position of fucosebound to the 6-position of N-acetylglucosamine at the reducing end ofthe sugar chain (see, e.g., U.S. Application No. 2010/0092997).Particular embodiments may include IgG Fc region that is glycosylatedwith at least one galactose moiety connected to a respective terminalsialic acid moiety by an α-2,6 linkage, optionally where the Fc regionhas a higher anti-inflammatory activity relative to a corresponding,wild-type Fc region (see U.S. Application No. 2008/0206246). Certain ofthese and related altered glycosylation approaches have generatedsubstantial enhancements of the capacity of Fc regions to selectivelybind FcRs such as FcγRIII, to mediate ADCC, and to alter otherproperties of Fc regions, as described herein.

Certain variant, fragment, hybrid, or otherwise modified Fc regions mayhave altered binding to one or more FcRs, relative to a corresponding,wild-type Fc sequence (e.g., same species, same Ig class, same Igsubclass). For instance, such Fc regions may have increased binding toone or more of Fcγ receptors, Fca receptors, Fcεreceptors, and/or theneonatal Fc receptor, relative to a corresponding, wild-type Fcsequence. In other embodiments, variant, fragment, hybrid, or modifiedFc regions may have decreased binding to one or more of Fcγ receptors,Fca receptors, Fcεreceptors, and/or the neonatal Fc receptor, relativeto a corresponding, wild-type Fc sequence. Specific FcRs are describedelsewhere herein.

Specific examples of Fc variants having altered (e.g., increased,decreased) FcR binding can be found, for example, in U.S. Pat. Nos.5,624,821 and 7,425,619; U.S. Application Nos. 2009/0017023,2009/0010921, and 2010/0203046; and WO 2000/42072 and WO 2004/016750.Certain examples include human Fc regions having a one or moresubstitutions at position 298, 333, and/or 334, for example, S298A,E333A, and/or K334A (based on the numbering of the EU index of Kabat etal.), which have been shown to increase binding to the activatingreceptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb.These mutations can be combined to obtain double and triple mutationvariants that have further improvements in binding to FcRs. Certainembodiments include a S298A/E333A/K334A triple mutant, which hasincreased binding to FcγRIIIa, decreased binding to FcγRIIb, andincreased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604,2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See alsoengineered Fc glycoforms that have increased binding to FcRs, asdisclosed in Umana et al., supra; and U.S. Pat. No. 7,662,925. Someembodiments include Fc regions that comprise one or more substitutionsselected from 434S, 252Y/428L, 252Y/434S, and 428L/4345 (see U.S.Application Nos. 2009/0163699 and 20060173170), based on the EU index ofKabat et al.

Certain variant, fragment, hybrid, or modified Fc regions may havealtered effector functions, relative to a corresponding, wild-type Fcsequence. For example, such Fc regions may have increased complementfixation or activation, increased C1q binding affinity, increasedCDC-related activity, increased ADCC-related activity, and/or increasedADCP-related activity, relative to a corresponding, wild-type Fcsequence. In other embodiments, such Fc regions may have decreasedcomplement fixation or activation, decreased C1q binding affinity,decreased CDC-related activity, decreased ADCC-related activity, and/ordecreased ADCP-related activity, relative to a corresponding, wild-typeFc sequence. As merely one illustrative example, an Fc region maycomprise a deletion or substitution in a complement-binding site, suchas a C1q-binding site, and/or a deletion or substitution in an ADCCsite. Examples of such deletions/substitutions are described, forexample, in U.S. Pat. No. 7,030,226. Many Fc effector functions, such asADCC, can be assayed according to routine techniques in the art. (see,e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Usefuleffector cells for such assays includes, but are not limited to, naturalkiller (NK) cells, macrophages, and other peripheral blood mononuclearcells (PBMC). Alternatively, or additionally, certain Fc effectorfunctions may be assessed in vivo, for example, by employing an animalmodel described in Clynes et al. PNAS. 95:652-656, 1998.

Certain variant hybrid, or modified Fc regions may have alteredstability or half-life relative to a corresponding, wild-type Fcsequence. In certain embodiments, such Fc regions may have increasedhalf-life relative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedhalf-life relative to a corresponding, wild-type Fc sequence. Half-lifecan be measured in vitro (e.g., under physiological conditions) or invivo, according to routine techniques in the art, such as radiolabeling,ELISA, or other methods. In vivo measurements of stability or half-lifecan be measured in one or more bodily fluids, including blood, serum,plasma, urine, or cerebrospinal fluid, or a given tissue, such as theliver, kidneys, muscle, central nervous system tissues, bone, etc. Asone example, modifications to an Fc region that alter its ability tobind the FcRn can alter its half-life in vivo. Assays for measuring thein vivo pharmacokinetic properties (e.g., in vivo mean eliminationhalf-life) and non-limiting examples of Fc modifications that alter itsbinding to the FcRn are described, for example, in U.S. Pat. Nos.7,217,797 and 7,732,570; and U.S. Application Nos. US 2010/0143254 and2010/0143254.

Additional non-limiting examples of modifications to alter stability orhalf-life include substitutions/deletions at one or more of amino acidresidues selected from 251-256, 285-290, and 308-314 in the CH₂ domain,and 385-389 and 428-436 in the CH₃ domain, according to the numberingsystem of Kabat et al. See U.S. Application No. 2003/0190311. Specificexamples include substitution with leucine at position 251, substitutionwith tyrosine, tryptophan or phenylalanine at position 252, substitutionwith threonine or serine at position 254, substitution with arginine atposition 255, substitution with glutamine, arginine, serine, threonine,or glutamate at position 256, substitution with threonine at position308, substitution with proline at position 309, substitution with serineat position 311, substitution with aspartate at position 312,substitution with leucine at position 314, substitution with arginine,aspartate or serine at position 385, substitution with threonine orproline at position 386, substitution with arginine or proline atposition 387, substitution with proline, asparagine or serine atposition 389, substitution with methionine or threonine at position 428,substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,and/or substitution with histidine, tyrosine, arginine or threonine atposition 436, including any combination thereof. Such modificationsoptionally increase affinity of the Fc region for the FcRn and therebyincrease half-life, relative to a corresponding, wild-type Fc region.

Certain variant hybrid, or modified Fc regions may have alteredsolubility relative to a corresponding, wild-type Fc sequence. Incertain embodiments, such Fc regions may have increased solubilityrelative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedsolubility relative to a corresponding, wild-type Fc sequence.Solubility can be measured, for example, in vitro (e.g., underphysiological conditions) according to routine techniques in the art.Exemplary solubility measurements are described elsewhere herein.

Additional examples of variants include IgG Fc regions havingconservative or non-conservative substitutions (as described elsewhereherein) at one or more of positions 250, 314, or 428 of the heavy chain,or in any combination thereof, such as at positions 250 and 428, or atpositions 250 and 314, or at positions 314 and 428, or at positions 250,314, and 428 (see, e.g., U.S. Application No. 2011/0183412). In specificembodiments, the residue at position 250 is substituted with glutamicacid or glutamine, and/or the residue at position 428 is substitutedwith leucine or phenylalanine. As another illustrative example of an IgGFc variant, any one or more of the amino acid residues at positions 214to 238, 297 to 299, 318 to 322, and/or 327 to 331 may be used as asuitable target for modification (e.g., conservative or non-conservativesubstitution, deletion). In particular embodiments, the IgG Fc variantCH₂ domain contains amino acid substitutions at positions 228, 234, 235,and/or 331 (e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) toattenuate the effector functions of the Fc region (see U.S. Pat. No.7,030,226). Here, the numbering of the residues in the heavy chain isthat of the EU index (see Kabat et al., “Sequences of Proteins ofImmunological Interest,” 5^(th) Ed., National Institutes of Health,Bethesda, Md. (1991)). Certain of these and related embodiments havealtered (e.g., increased, decreased) FcRn binding and/or serumhalf-life, optionally without reduced effector functions such as ADCC orCDC-related activities.

Additional examples include variant Fc regions that comprise one or moreamino acid substitutions at positions 279, 341, 343 or 373 of awild-type Fc region, or any combination thereof (see, e.g., U.S.Application No. 2007/0224188). The wild-type amino acid residues atthese positions for human IgG are valine (279), glycine (341), proline(343) and tyrosine (373). The substation(s) can be conservative ornon-conservative, or can include non-naturally occurring amino acids ormimetics, as described herein. Alone or in combination with thesesubstitutions, certain embodiments may also employ a variant Fc regionthat comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions selected from the following: 235G, 235R, 236F, 236R, 236Y,237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L,245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W,247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I,251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P,254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V,256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K,265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R,279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R,279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M,283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G,292I, 292L, 293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L,315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L,332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L,339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I,341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y,343A, 343D, 343E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q,343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I,373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E,376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T,376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D,380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P,382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H,424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G,430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V,430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W,439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M,440Q, 440T, 440V or 442K. As above, the numbering of the residues in theheavy chain is that of the EU index (see Kabat et al., supra). Suchvariant Fc regions typically confer an altered effector function oraltered serum half-life upon HRS polypeptide to which the variant Fcregion is operably attached. Preferably the altered effector function isan increase in ADCC, a decrease in ADCC, an increase in CDC, a decreasein CDC, an increase in C1q binding affinity, a decrease in C1q bindingaffinity, an increase in FcR (preferably FcRn) binding affinity or adecrease in FcR (preferably FcRn) binding affinity as compared to acorresponding Fc region that lacks such amino acid substitution(s).

Additional examples include variant Fc regions that comprise an aminoacid substitution at one or more of position(s) 221, 222, 224, 227, 228,230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288,290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335 336 and/or 428 (see, e.g., U.S. Pat. No. 7,662,925). In specificembodiments, the variant Fc region comprises at least one amino acidsubstitution selected from the group consisting of: P230A, E233D, L234E,L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q,S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T,K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V,L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R,and T335Y. In other specific embodiments, the variant Fc regioncomprises at least one amino acid substitution selected from the groupconsisting of: V264I, F243L/V264I, L328M, I332E, L328M/I332E,V264I/I332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y, I332D,L328I/I332E, L328Q/I332E, V264T, V240I, V266I, S239D, S239D/I332D,S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D, S239E/I332N,S239E/I332Q, S239N/I332D, S239N/I332E, S239Q/I332D, A330Y/I332E,V264I/A330Y/I332E, A330L/I332E, V264I/A330L/I332E, L234E, L234Y, L234I,L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T,L328D/I332E, L328V/I332E, L328T/I332E, L328I/I332E, S239EN264I/I332E,S239QN264I/I332E, S239EN264I/A330Y/I332E, S239D/A330Y/I332E,S239N/A330Y/I332E, S239D/A330L/I332E, S239N/A330L/I332E,V264I/S298A/I332E, S239D/S298A/I332E, S239N/S298A/I332E,S239DN264I/I332E, S239DN264I/S298A/I332E, S239DN264I/A330L/I332E,S239D/I332E/A330I, P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R,K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V,K326I, K326T, T335D, T335R, T335Y, V240I/V266I, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332EN240I, S239D/A330Y/I332EN264T,S239D/A330Y/I332E/K326E, and S239D/A330Y/I332E/K326T, In more specificembodiments, the variant Fc region comprises a series of substitutionsselected from the group consisting of: N297D/I332E,F241Y/F243Y/V262T/V264T/N297D/I332E, S239D/N297D/I332E,S239E/N297D/I332E, S239D/D265Y/N297D/I332E, S239D/D265H/N297D/I332E,V264E/N297D/I332E, Y296N/N297D/I332E, N297D/A330Y/I332E,S239D/D265V/N297D/I332E, S239D/D265I/N297D/I332E, andN297D/S298A/A330Y/I332E. In specific embodiments, the variant Fc regioncomprises an amino acid substitution at position 332 (using thenumbering of the EU index, Kabat et al., supra). Examples ofsubstitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L,332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W and 332Y. Thenumbering of the residues in the Fc region is that of the EU index ofKabat et al. Among other properties described herein, such variant Fcregions may have increased affinity for an FcγR, increased stability,and/or increased solubility, relative to a corresponding, wild-type Fcregion.

Further examples include variant Fc regions that comprise one or more ofthe following amino acid substitutions: 224N/Y, 225A, 228L, 230S, 239P,240A, 241L, 243S/L/G/H/I, 244L, 246E, 247L/A, 252T, 254T/P, 258K, 261Y,265V, 266A, 267G/N, 268N, 269K/G, 273A, 276D, 278H, 279M, 280N, 283G,285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A,306I/F, 311R, 312N, 315D/K/S, 320R, 322E, 323A, 324T, 325S, 326E/R,332T, 333D/G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S,352A, 354A, 355W, 356G, 358T, 361D/Y, 362L, 364C, 365Q/P, 370R, 372L,377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R,411I, 412A, 414M, 421S, 422I, 426F/P, 428T, 430K, 431S, 432P, 433P,438L, 439E/R, 440G, 441F, 442T, 445R, 446A, 447E, optionally where thevariant has altered recognition of an Fc ligand and/or altered effectorfunction compared with a parent Fc polypeptide, and wherein thenumbering of the residues is that of the EU index as in Kabat et al.Specific examples of these and related embodiments include variant Fcregions that comprise or consist of the following sets of substitutions:(1) N276D, R292Q, V305A, I377V, T394A, V412A and K439E; (2) P244L,K246E, D399G and K409R; (3) 5304G, K320R, S324T, K326E and M358T; (4)F243S, P247L, D265V, V266A, S383N and T411I; (5) H224N, F243L, T393A andH433P; (6) V240A, S267G, G341E and E356G; (7) M252T, P291L, P352A,R355W, N390D, 5408G, S426F and A431S; (8) P228L, T289A, L365Q, N389S and5440G; (9) F241L, V273A, K340Q and L441F; (10) F241L, T299A, I332T andM428T; (11) E269K, Y300H, Q342R, V422I and G446A; (12) T225A, R301c,5304G, D312N, N315D, L351S and N421S; (13) S254T, L306I, K326R andQ362L; (14) H224Y, P230S, V323A, E333D, K338R and S364C; (15) T335I,K414M and P445R; (16) T335I and K414M; (17) P247A, E258K, D280N, K288R,N297D, T299A, K322E, Q342R, S354A and L365P; (18) H268N, V279M, A339T,N361D and S426P; (19) C261Y, K290E, L306F, Q311R, E333G and Q438L; (20)E283G, N315K, E333G, R344Q, L365P and S442T; (21) Q347R, N361Y andK439R; (22) S239P, S254P, S267N, H285R, N3155, F372L, A378T, N390D,Y391C, F404S, E430K, L432P and K447E; and (23) E269G, Y278H, N325S andK370R, wherein the numbering of the residues is that of the EU index asin Kabat et al. (see, e.g., U.S. Application No. 2010/0184959).

Another specific example of an Fc variant comprises the sequence of SEQID NO:155, wherein Xaa at position 1 is Ala or absent; Xaa at position16 is Pro or Glu; Xaa at position 17 is Phe, Val, or Ala; Xaa atposition 18 is Leu, Glu, or Ala; Xaa at position 80 is Asn or Ala;and/or Xaa at position 230 is Lys or is absent (see, e.g., U.S.Application No. 2007/0253966). Certain of these Fc regions, and relatedHRS-Fc conjugates, have increased half-life, reduced effector activity,and/or are significantly less immunogenic than wild-type Fc sequences.

Variant Fc regions can also have one or more mutated hinge regions, asdescribed, for example, in U.S. Application No. 2003/0118592. Forinstance, one or more cysteines in a hinge region can be deleted orsubstituted with a different amino acid. The mutated hinge region cancomprise no cysteine residues, or it can comprise 1, 2, or 3 fewercysteine residues than a corresponding, wild-type hinge region. In someembodiments, an Fc region having a mutated hinge region of this typeexhibits a reduced ability to dimerize, relative to a wild-type Ig hingeregion.

As noted above, HRS-Fc conjugates such as HRS-Fc fusion proteinstypically have altered (e.g., improved, increased, decreased)pharmacokinetic properties relative to corresponding HRS polypeptides.Examples of pharmacokinetic properties include stability or half-life,bioavailability (the fraction of a drug that is absorbed), tissuedistribution, volume of distribution (apparent volume in which a drug isdistributed immediately after it has been injected intravenously andequilibrated between plasma and the surrounding tissues), concentration(initial or steady-state concentration of drug in plasma), eliminationrate constant (rate at which drugs are removed from the body),elimination rate (rate of infusion required to balance elimination),area under the curve (AUC or exposure; integral of theconcentration-time curve, after a single dose or in steady state),clearance (volume of plasma cleared of the drug per unit time), C_(max)(peak plasma concentration of a drug after oral administration), t_(max)(time to reach C_(max)), C_(min) (lowest concentration that a drugreaches before the next dose is administered), and fluctuation (peaktrough fluctuation within one dosing interval at steady state). In someaspects, these improved properties are achieved without significantlyaltering the secondary structure and/or reducing the non-canonicalbiological activity of the HRS polypeptide. Indeed, some HRS-Fcconjugates have increased non-canonical biological activity.

Hence, in some embodiments, the HRS-Fc conjugate or HRS-Fc fusionpolypeptide has a plasma or sera pharmacokinetic AUC profile at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50,100, 200, 300, 400, or 500-fold greater than a corresponding unmodifiedor differently modified HRS polypeptide when administered to a mammalunder the same or comparable conditions. In certain embodiments, theHRS-Fc conjugate or HRS-Fc fusion polypeptide has a stability (e.g., asmeasured by half-life) which is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater than acorresponding unmodified or differently modified HRS polypeptide whencompared under similar conditions at room temperature, for example, inPBS at pH 7.4 for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days, or 1, 2, 3, 4 weeks or so.

In particular embodiments, a HRS-Fc conjugate or HRS-Fc fusionpolypeptide has a biological half life at pH 7.4, 25° C., e.g., aphysiological pH, human body temperature (e.g., in vivo, in serum, in agiven tissue, in a given species such as rat, mouse, monkey, or human),of about or at least about 30 minutes, about 1 hour, about 2 hour, about3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours,about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours,about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90hours, about 96 hours, about 120 hours, or about 144 hours or more orany intervening half-life.

In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusionpolypeptide has greater bioavailability after subcutaneous (SC)administration compared to a corresponding unmodified HRS-polypeptide.In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusionpolypeptide has at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 100%, or morebioavailability compared to the corresponding unmodified HRSpolypeptide.

In certain embodiments, the HRS-Fc fusion polypeptide has substantiallythe same secondary structure as a corresponding unmodified ordifferently modified HRS polypeptide, as determined via UV circulardichroism analysis. In certain embodiments, the HRS-Fc fusionpolypeptide has substantially the same activity of a correspondingunmodified or differently modified HRS polypeptide in an assay ofanti-inflammatory activity. In other embodiments, the HRS-Fc fusionpolypeptide has greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20-fold the activity of a correspondingunmodified or differently modified HRS polypeptide in an assay ofanti-inflammatory activity.

Peptide Linkers

In certain embodiments, a peptide linker sequence may be employed toseparate the HRS polypeptide(s) and the Fc region(s) by a distancesufficient to ensure that each polypeptide folds into its desiredsecondary and tertiary structures. Such a peptide linker sequence can beincorporated into the fusion protein using standard techniques wellknown in the art.

Certain peptide linker sequences may be chosen based on the followingexemplary factors: (1) their ability to adopt a flexible extendedconformation; (2) their inability to adopt a secondary structure thatcould interact with functional epitopes on the first and secondpolypeptides; (3) their physiological stability; and (4) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes, or other features. See, e.g., George and Heringa, JProtein Eng. 15:871-879, 2002.

The linker sequence may generally be from 1 to about 200 amino acids inlength. Particular linkers can have an overall amino acid length ofabout 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 ormore amino acids.

A peptide linker may employ any one or more naturally-occurring aminoacids, non-naturally occurring amino acid(s), amino acid analogs, and/oramino acid mimetics as described elsewhere herein and known in the art.Certain amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphyet al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and4,751,180. Particular peptide linker sequences contain Gly, Ser, and/orAsn residues. Other near neutral amino acids, such as Thr and Ala mayalso be employed in the peptide linker sequence, if desired.

Certain exemplary linkers include Gly, Ser and/or Asn-containinglinkers, as follows: [G]_(x), [S]_(x), [GS]_(x), [GGS]_(x), [GSS]_(x),[GSGS]_(x) (SEQ ID NO:200), [GGSG]_(x) (SEQ ID NO:201), [GGGS]_(x) (SEQID NO:202), [GGGGS]_(x) (SEQ ID NO:203), [GN]_(x), [GGN]_(x), [GNN]_(x),[GNGN]_(x) (SEQ ID NO: 204), [GGNG]_(x) (SEQ ID NO:205), [GGGN]_(x) (SEQID NO:206), [GGGGN]_(x) (SEQ ID NO:207) linkers, where x is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more.Other combinations of these and related amino acids will be apparent topersons skilled in the art.

Additional examples of linker peptides include, but are not limited tothe following amino acid sequences:Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ IDNO:208);Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQID NO:209);Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQID NO:210);Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(SEQID NO:211); andAsn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO:212).

Further non-limiting examples of linker peptides include DGGGS (SEQ IDNO:213); TGEKP (SEQ ID NO:214) (see, e.g., Liu et al., PNAS.94:5525-5530, 1997); GGRR (SEQ ID NO:215) (Pomerantz et al. 1995);(GGGGS)_(n) (SEQ ID NO:203) (Kim et al., PNAS. 93:1156-1160, 1996);EGKSSGSGSESKVD (SEQ ID NO:216) (Chaudhary et al., PNAS. 87:1066-1070,1990); KESGSVSSEQLAQFRSLD (SEQ ID NO:217) (Bird et al., Science.242:423-426, 1988), GGRRGGGS (SEQ ID NO:218); LRQRDGERP (SEQ ID NO:219);LRQKDGGGSERP (SEQ ID NO:220); LRQKd(GGGS)₂ ERP (SEQ ID NO:221). Inspecific embodiments, the linker sequence comprises a Gly3 linkersequence, which includes three glycine residues. In particularembodiments, flexible linkers can be rationally designed using acomputer program capable of modeling both DNA-binding sites and thepeptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; andPNAS. 91:11099-11103, 1994) or by phage display methods.

The peptide linkers may be physiologically stable or may include areleasable linker such as a physiologically degradable or enzymaticallycleavable linker (e.g., proteolytically cleavable linker). In certainembodiments, one or more releasable linkers can result in a shorterhalf-life and more rapid clearance of the conjugate. These and relatedembodiments can be used, for example, to enhance the solubility andblood circulation lifetime of HRS polypeptides in the bloodstream, whilealso delivering a HRS polypeptide into the bloodstream that, subsequentto linker degradation, is substantially free of the Fc region(s). Theseaspects are especially useful in those cases where HRS polypeptides,when permanently conjugated to an Fc region, demonstrate reducedactivity. By using the linkers as provided herein, such HRS polypeptidescan maintain their therapeutic activity when in conjugated form. Asanother example, a large and relatively inert HRS-Fc conjugatepolypeptide may be administered, which is then degraded in vivo (via thedegradable linker) to generate a bioactive HRS polypeptide possessing aportion of the Fc region or lacking the Fc region entirely. In these andother ways, the properties of the HRS-Fc conjugate polypeptide can bemore effectively tailored to balance the bioactivity and circulatinghalf-life of the HRS polypeptide over time.

In particular embodiments, the linker peptide comprises an autocatalyticor self-cleaving peptide cleavage site. In a particular embodiment,self-cleaving peptides include those polypeptide sequences obtained frompotyvirus and cardiovirus 2A peptides, FMDV (foot-and-mouth diseasevirus), equine rhinitis A virus, Thosea asigna virus and porcineteschovirus. In certain embodiments, the self-cleaving polypeptide sitecomprises a 2A or 2A-like site, sequence or domain (Donnelly et al., J.Gen. Virol. 82:1027-1041, 2001). Exemplary 2A sites include thefollowing sequences: LLNFDLLKLAGDVESNPGP (SEQ ID NO:222);TLNFDLLKLAGDVESNPGP (SEQ ID NO: 223); LLKLAGDVESNPGP (SEQ ID NO:224);NFDLLKLAGDVESNPGP (SEQ ID NO:225); QLLNFDLLKLAGDVESNPGP (SEQ ID NO:226);APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:227);VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO:228);LNFDLLKLAGDVESNPGP (SEQ ID NO:229);LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:230); andEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:231). In one embodiment,the autocatalytic peptide cleavage site comprises a translational 2Asignal sequence, such as, e.g., the 2A region of the aphthovirusfoot-and-mouth disease virus (FMDV) polyprotein, which is an18 aminoacid sequence. Additional examples of 2A-like sequences that may be usedinclude insect virus polyproteins, the NS34 protein of type Crotaviruses, and repeated sequences in Trypanosoma spp., as described,for example, in Donnelly et al., Journal of General Virology.82:1027-1041, 2001.

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., Ryan et al., J. Gener. Virol.78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus NIa proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.Due to its high cleavage stringency, TEV (tobacco etch virus) proteasecleavage sites are included in some embodiments, e.g., EXXYXQ(G/S) (SEQID NO:232), for example, ENLYFQG (SEQ ID NO:233) and ENLYFQS (SEQ IDNO:234), wherein X represents any amino acid (cleavage by TEV occursbetween Q and G or Q and S).

Further examples of enzymatically degradable linkers suitable for use inparticular embodiments of the present invention include, but are notlimited to: an amino acid sequence cleaved by a serine protease such asthrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin.Illustrative examples of thrombin-cleavable amino acid sequencesinclude, but are not limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO:235),-Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO: 236),-Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:237), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQID NO: 238), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-.Illustrative examples of elastase-cleavable amino acid sequencesinclude, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val-(SEQ IDNO:239), -Ala-Ala-Pro-Leu-(SEQ ID NO:240), -Ala-Ala-Pro-Phe-(SEQ IDNO:241), -Ala-Ala-Pro-Ala-(SEQ ID NO:242), and -Ala-Tyr-Leu-Val-(SEQ IDNO:243).

Enzymatically degradable linkers also include amino acid sequences thatcan be cleaved by a matrix metalloproteinase such as collagenase,stromelysin, and gelatinase. Illustrative examples of matrixmetalloproteinase-cleavable amino acid sequences include, but are notlimited to: -Gly-Pro-Y-Gly-Pro-Z-(SEQ ID NO:244), -Gly-Pro-,Leu-Gly-Pro-Z-(SEQ ID NO:245), -Gly-Pro-Ile-Gly-Pro-Z-(SEQ ID NO:246),and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO:247), where Y and Z are amino acids.Illustrative examples of collagenase-cleavable amino acid sequencesinclude, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ IDNO:248), -Pro-Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO:249),-Pro-Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO:250),-Pro-Leu-Gly-Cys(Me)-His-(SEQ ID NO:251), -Pro-Leu-Gly-Leu-Tyr-Ala-(SEQID NO:252), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQ ID NO:253), and-Pro-Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO:254), where Z is an amino acid.An illustrative example of a stromelysin-cleavable amino acid sequenceis -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO:255); and an example of agelatinase-cleavable amino acid sequence is-Pro-Leu-Gly-Met-Tyr-Ser-Arg-(SEQ ID NO:256).

Enzymatically degradable linkers suitable for use in particularembodiments of the present invention also include amino acid sequencesthat can be cleaved by an angiotensin converting enzyme, such as, forexample, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO:257), and-Gly-Ser-Asp-Lys-Pro-(SEQ ID NO:258).

Enzymatically degradable linkers suitable for use in particularembodiments of the present invention also include amino acid sequencesthat can be degraded by cathepsin B, such as, for example, Val-Cit,Ala-Leu-Ala-Leu-(SEQ ID NO:259), Gly-Phe-Leu-Gly-(SEQ ID NO:260) andPhe-Lys.

In particular embodiments, a releasable linker has a half life at pH7.4, 25° C., e.g., a physiological pH, human body temperature (e.g., invivo, in serum, in a given tissue), of about 30 minutes, about 1 hour,about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours,about 48 hours, about 72 hours, or about 96 hours or more or anyintervening half-life. One having skill in the art would appreciate thatthe half life of a HRS-Fc conjugate polypeptide can be finely tailoredby using a particular releasable linker.

In certain embodiments, however, any one or more of the peptide linkersare optional. For instance, linker sequences may not required when thefirst and second polypeptides have non-essential N-terminal and/orC-terminal amino acid regions that can be used to separate thefunctional domains and prevent steric interference.

Methods for Use

Embodiments of the present invention relate to the discovery that Fcregion-histidyl-tRNA synthetase (HRS-Fc) conjugate polypeptides, andfragments and variants thereof, offer improved methods of modulatinginflammatory responses in a variety of useful ways, both in vitro and invivo. The compositions of the invention may thus be useful asimmunomodulators for treating a broad range of pro-inflammatory,inflammatory, and/or autoimmune indications, including inflammatoryresponses, chronic inflammation, acute inflammation, and immunediseases, by modulating the cells that mediate, either directly orindirectly, such inflammatory and/or autoimmune diseases, conditions anddisorders. The utility of the compositions of the invention asimmunomodulators can be monitored using any of a number of known andavailable techniques in the art including, for example, migration assays(e.g., using leukocytes or lymphocytes), cytokine production assays, orcell viability or cell differentiation assays (e.g., using B-cells,T-cells, monocytes or NK cells).

“Inflammation” refers generally to the biological response of tissues toharmful stimuli, such as pathogens, damaged cells (e.g., wounds), andirritants. The term “inflammatory response” refers to the specificmechanisms by which inflammation is achieved and regulated, including,merely by way of illustration, immune cell activation or migration,migration, autoimmunity and autoimmune disease, cytokine production,vasodilation, including kinin release, fibrinolysis, and coagulation,among others described herein and known in the art. Ideally,inflammation is a protective attempt by the body to both remove theinjurious stimuli and initiate the healing process for the affectedtissue or tissues. In the absence of inflammation, wounds and infectionswould never heal, creating a situation in which progressive destructionof the tissue would threaten survival. On the other hand, excessive orchronic inflammation may associate with a variety of diseases, such ashay fever, atherosclerosis, and rheumatoid arthritis, among othersdescribed herein and known in the art.

Clinical signs of chronic inflammation are dependent upon duration ofthe illness, inflammatory lesions, cause and anatomical area affected,(see, e.g., Kumar et al., Robbins Basic Pathology-8 ft Ed., 2009Elsevier, London; Miller, L M, Pathology Lecture Notes, AtlanticVeterinary College, Charlottetown, PEI, Canada). Chronic inflammation isassociated with a variety of pathological conditions or diseases,including, for example, allergies, Alzheimer's disease, anemia, aorticvalve stenosis, arthritis such as rheumatoid arthritis andosteoarthritis, cancer, congestive heart failure, fibromyalgia,fibrosis, heart attack, kidney failure, lupus, pancreatitis, stroke,surgical complications, inflammatory lung disease, inflammatory boweldiseases including Crohn's disease (CD) and ulcerative colitis (UC),atherosclerosis, neurological disorders, diabetes, metabolic disorders,obesity, and psoriasis, among others described herein and known in theart. Many other chronic diseases may also include an inflammatorycomponent, and thus may be treated with the HRS-Fc conjugates of theinvention including, for example, muscular dystrophies andrhabdomyolysis. Hence, HRS-Fc conjugates may be used to treat or managechronic inflammation, modulate any of one or more of the individualchronic inflammatory responses, or treat any one or more diseases orconditions associated with chronic inflammation.

Certain specific inflammatory responses include cytokine production andactivity, and related pathways. For instance, certain exemplaryembodiments relate to modulating cell-signaling through nuclearfactor-kB (NF-kB), such as by increasing the downstream activities ofthis transcription factor. In certain instances, increases in NF-kBactivity can lead to increases in cytokine signaling or activity, suchas pro-inflammatory cytokines (e.g., TNF-alpha or beta), andanti-inflammatory cytokines (e.g., IL-10).

Criteria for assessing the signs and symptoms of inflammatory and otherconditions, including for purposes of making differential diagnosis andalso for monitoring treatments such as determining whether atherapeutically effective dose has been administered in the course oftreatment, e.g., by determining improvement according to acceptedclinical criteria, will be apparent to those skilled in the art and areexemplified by the teachings of e.g., Berkow et al., eds., The MerckManual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al.,eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics,10th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins,Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brown and Co.,Boston, (1985); Osolci al., eds., Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Co., Easton, Pa. (1990); Katzung, Basicand Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992).

Also included are methods of modulating an immune response, such as aninnate or adaptive immune response via the use of any of the HRS-Fcconjugates described herein. As used herein, the term “immune response”includes a measurable or observable reaction to an antigen, vaccinecomposition, or immunomodulatory molecule mediated by one or more cellsof the immune system. An immune response typically begins with anantigen or immunomodulatory molecule binding to an immune system cell. Areaction to an antigen or immunomodulatory molecule may be mediated bymany cell types, including a cell that initially binds to an antigen orimmunomodulatory molecule and cells that participate in mediating aninnate, humoral, cell-mediated immune response.

Also included are methods of treating immune diseases. Illustrativeimmune system diseases, disorders or conditions that may be treatedaccording to the present invention include, but are not limited to,primary immunodeficiencies, immune-mediated thrombocytopenia, Kawasakisyndrome, bone marrow transplant (for example, recent bone marrowtransplant in adults or children), chronic B cell lymphocytic leukemia,HIV infection (for example, adult or pediatric HIV infection), chronicinflammatory demyelinating polyneuropathy, post-transfusion purpura, andthe like.

Additionally, further diseases, disorders and conditions which may betreated with any of the HRS-Fc conjugates described herein includeGuillain-Barre syndrome, anemia (for example, anemia associated withparvovirus B19, patients with stable multiple myeloma who are at highrisk for infection (for example, recurrent infection), autoimmunehemolytic anemia (for example, warm-type autoimmune hemolytic anemia),thrombocytopenia (for example, neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (for example,cytomegalovirus (CMV)-negative recipients of CMV-positive organs),hypogammaglobulinemia (for example, hypogammaglobulinemic neonates withrisk factor for infection or morbidity), epilepsy (for example,intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis(for example, decompensation in myasthenia gravis), dermatomyositis, andpolymyositis.

Further autoimmune diseases, disorders and conditions which may betreated with any of the HRS-Fc conjugates described herein include butare not limited to, autoimmune hemolytic anemia, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (forexample, IgA nephropathy), multiple sclerosis, neuritis, uveitisophthalmia, polyendochnopathies, purpura (for example, Henloch-Scoenleinpurpura), Reiter's disease, stiff-man syndrome, autoimmune pulmonaryinflammation, Guillain-Barre Syndrome, insulin dependent diabetesmellitus, and autoimmune inflammatory eye disease.

Additional autoimmune diseases, disorders or conditions which may betreated with any of the HRS-Fc conjugates described herein include, butare not limited to, autoimmune thyroiditis; hypothyroidism, includingHashimoto's thyroiditis and thyroiditis characterized, for example, bycell-mediated and humoral thyroid cytotoxicity; SLE (which is oftencharacterized, for example, by circulating and locally generated immunecomplexes); Goodpasture's syndrome (which is often characterized, forexample, by anti-basement membrane antibodies); pemphigus (which isoften characterized, for example, by epidermal acantholytic antibodies);receptor autoimmunities such as, for example, Graves' disease (which isoften characterized, for example, by antibodies to a thyroid stimulatinghormone receptor; myasthenia gravis, which is often characterized, forexample, by acetylcholine receptor antibodies); insulin resistance(which is often characterized, for example, by insulin receptorantibodies); autoimmune hemolytic anemia (which is often characterized,for example, by phagocytosis of antibody-sensitized red blood cells);and autoimmune thrombocytopenic purpura (which is often characterized,for example, by phagocytosis of antibody-sensitized platelets).

Further autoimmune diseases, disorders or conditions which may betreated with any of the HRS-Fc conjugates described herein include, butare not limited to, rheumatoid arthritis (which is often characterized,for example, by immune complexes in joints); scleroderma withanti-collagen antibodies (which is often characterized, for example, bynucleolar and other nuclear antibodies); mixed connective tissuedisease, (which is often characterized, for example, by antibodies toextractable nuclear antigens, for example, ribonucleoprotein);polymyositis/dermatomyositis (which is often characterized, for example,by nonhistone anti-nuclear antibodies); pernicious anemia (which isoften characterized, for example, by antiparietal cell, antimicrosome,and anti-intrinsic factor antibodies); idiopathic Addison's disease(which is often characterized, for example, by humoral and cell-mediatedadrenal cytotoxicity); infertility (which is often characterized, forexample, by antispennatozoal antibodies); glomerulonephritis (which isoften characterized, for example, by glomerular basement membraneantibodies or immune complexes); by primary glomerulonephritis, by IgAnephropathy; bullous pemphigoid (which is often characterized, forexample, by IgG and complement in the basement membrane); Sjogren'ssyndrome (which is often characterized, for example, by multiple tissueantibodies and/or the specific nonhistone antinuclear antibody (SS-B));diabetes mellitus (which is often characterized, for example, bycell-mediated and humoral islet cell antibodies); and adrenergic drugresistance, including adrenergic drug resistance with asthma or cysticfibrosis (which is often characterized, for example, by beta-adrenergicreceptor antibodies).

Still further autoimmune diseases, disorders or conditions which may betreated with any of the HRS-Fc conjugates described herein include, butare not limited to chronic active hepatitis (which is oftencharacterized, for example by smooth muscle antibodies); primary biliarycirrhosis (which is often characterized, for example, byanti-mitochondrial antibodies); other endocrine gland failure (which ischaracterized, for example, by specific tissue antibodies in somecases); vitiligo (which is often characterized, for example, byanti-melanocyte antibodies); vasculitis (which is often characterized,for example, by immunoglobulin and complement in vessel walls and/or lowserum complement); post-myocardial infarction conditions (which areoften characterized, for example, by anti-myocardial antibodies);cardiotomy syndrome (which is often characterized, for example, byanti-myocardial antibodies); urticaria (which is often characterized,for example, by IgG and IgM antibodies to IgE); atopic dermatitis (whichis often characterized, for example, by IgG and IgM antibodies to IgE);asthma (which is often characterized, for example, by IgG and IgMantibodies to IgE); inflammatory myopathies; and other inflammatory,granulomatous, degenerative, and atrophic disorders.

Additional diseases and disorders which may be treated with any of theHRS-Fc conjugates described herein include those that result from orassociate with an imbalance of Th17 or other Th cell subtypes. Examplesinclude psoriasis, psoriatic arthritis, atopic dermatitis (eczema), Baloconcentric sclerosis, Schilder's diffuse sclerosis, Marburg MS, IBD,Crohn's, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behçet's disease, indeterminatecolitis, asthma, autoimmune myocarditis, endometriosis, Adult onsetStill's disorder (AOSD), Henoch-Schonlein purpura (HSP),Vogt-Koyanagi-Harada (VKH), periodontal disease, organ transplantationfailure, graft versus host disease, and Devic's disease (neuromyelitisoptica).

In some aspects, the present invention includes a method of reducingmuscle or lung inflammation associated with an autoimmune diseasecomprising administering to a subject in need thereof a compositioncomprising any of the HRS-Fc conjugates described herein. Exemplarymuscular inflammatory diseases and disorders include musculardystrophies, exercise-induced muscle inflammation, inflammationassociated with muscle injury or surgery, rhabdomyolysis, and relateddiseases and disorders as described herein.

Also included are methods of treating a disease associated with anautoantibody comprising administering to a subject in need thereof atherapeutic composition comprising any of the HRS-Fc conjugatesdescribed herein, wherein the HRS polypeptide comprises at least oneepitope specifically recognized by the autoantibody.

Certain embodiments include methods of inducing tolerance to ahistidyl-tRNA synthetase (HisRS) antigen, said method comprisingadministering to a subject a composition comprising any of the HRS-Fcconjugates described herein, wherein the HRS polypeptide comprises atleast one epitope specifically recognized by the autoantibody, andwherein administration of the composition causes tolerization to theautoantigen.

Also included are methods for eliminating a set or subset of T cellsinvolved in an autoimmune response to a histidyl tRNA synthetase (HisRS)autoantigen, the method comprising administering to a subject acomposition comprising any of the HRS-Fc conjugates described herein,wherein the HRS polypeptide comprises at least one epitope specificallyrecognized by the autoantibody, or auto-reactive T cell, and whereinadministration of the composition causes clonal deletion ofauto-reactive T-cells.

In another embodiment, the present invention includes a method forinducing anergy in T cells involved in an autoimmune response to ahistidyl tRNA synthetase (HRS) autoantigen, the method comprisingadministering to a subject a composition comprising any of the HRS-Fcconjugates described herein, wherein the HRS polypeptide comprises atleast one epitope specifically recognized by the autoantibody, or Tcell, and wherein administration of the composition causes functionalinactivation of the T cells involved in the autoimmune response.

In another embodiment, the present invention includes a replacementtherapy for treating a disease associated with an insufficiency ofhistidyl tRNA synthetase comprising administering to a subject in needthereof a therapeutic composition comprising any of the HRS-Fcconjugates described herein, wherein the HRS polypeptide functionallycompensates for the histidyl tRNA synthetase insufficiency.

In one aspect of this replacement therapy, the histidyl tRNA synthetaseinsufficiency is caused by the presence of anti-Jo-1 antibodies. In oneaspect of this replacement therapy, the histidyl tRNA synthetaseinsufficiency is caused by mutations in an endogenous histidyl tRNAsynthetase which modulate the activity, expression or cellulardistribution of the endogenous histidyl tRNA synthetase. In one aspectthe histidyl tRNA synthetase insufficiency is associated with Perraultsyndrome or Usher syndrome.

In any of these methods, the term “tolerance” refers to the sustainedreduction or absence of an immune response to a specific antigen in amammal, particularly a human. Tolerance is distinct from generalizedimmunosuppression, in which all, or all of a specific class of immunecells, such as B cell mediated immune responses, of an immune responsesare diminished, or eliminated. The development of tolerance may beroutinely monitored by the absence, or a decrease, in the concentrationof antibodies to HRS polypeptides in the serum of the host subject afteradministration, in single or successive doses of the treating HRS-Fcconjugate. The development of tolerance will typically be sufficient todecrease the symptoms of the autoimmune disease in the patient, forexample a patient may be sufficiently improved so as to maintain normalactivities in the absence, or in the presence of reduced amounts, ofgeneral immunosuppressants, e.g. corticosteroids.

In any of these methods, and compositions tolerance will typically besustained, meaning that it will have a duration of about one month,about two months, about three months, about 4 months, about 5 months, orabout 6 months or longer. Tolerance may result in selective B-cellanergy, or T-cell anergy or both.

In any of these methods, treatments and therapeutic compositions, theterm “a disease associated with autoantibodies specific for histidyltRNA synthetase” refers to any disease or disorder in which antibodiesto histidyl tRNA synthetase are detected, or detectable, irrespective ofwhether other autoantibodies are also detected, or thought to play arole in disease progression or cause. Methods for detecting antibodiesin patient samples may be carried out by any standard procedureincluding for example, by RIA, ELISA, by immunoprecipitation, bystaining of tissues or cells (including transfected cells), antigenmicroarrays, mass spec analysis, specific neutralization assays or oneof a number of other methods known in the art for identifying desiredantigen specificity. In some aspects, antibody specificity can befurther characterized by determining the ability of the antibodies toselectively bind to different splice variants and truncated orproteolytic forms of histidyl tRNA synthetase. A relatively well knownhuman auto-antibody to histidyl tRNA synthetase includes for exampleantibodies to Jo-1.

In some embodiments of any of the claimed methods, and compositions, theHRS polypeptide or HRS-Fc conjugate comprises an epitope from histidyltRNA synthetase which specifically cross reacts with a diseaseassociated auto-antibody to histidyl-tRNA synthetase. In someembodiments of any of the claimed methods, and compositions, the HRSpolypeptide or HRS-Fc conjugate comprises an epitope from histidyl tRNAsynthetase which specifically cross reacts with a disease associatedauto-reactive T cell to histidyl-tRNA synthetase. In some embodiments ofany of the claimed methods, and compositions, the HRS polypeptide orHRS-Fc conjugate comprises an epitope which specifically cross reactswith a disease associated auto-antibody to either another tRNAsynthetase, or to a non tRNA synthetase auto antibody.

In some embodiments of any of the claimed methods the HRS polypeptide orHRS-Fc conjugate comprises an immunodominant epitope which isspecifically recognized by the majority of antibodies from the sera of apatient with a disease associated with auto antibodies to histidyl-tRNAsynthetase. In some embodiments of any of the claimed methods the HRSpolypeptide or HRS-Fc conjugate comprises an immunodominant epitopewhich is specifically recognized by the majority of autoreactive T cellsfrom the sera of a patient with a disease associated with autoantibodies to histidyl-tRNA synthetase.

In some embodiments, the epitope is comprised within the WHEP domain ofthe HRS polypeptide (approximately amino acids 1-43 of SEQ ID NO:1); theaminoacylation domain (approximately amino acids 54-398 of SEQ ID NO:1);or the anticodon binding domain (approximately amino acids 406-501 ofSEQ ID NO:1) or any combination thereof.

In some embodiments, the HRS polypeptide does not comprise an epitopewhich specifically cross reacts with a disease associated auto-antibodyto histidyl-tRNA synthetase. In some embodiments, the HRS polypeptidedoes not significantly compete for disease associated auto-antibodybinding to histidyl-tRNA synthetase in a competitive ELISA up to aconcentration of about 1×10⁻⁷M. In some embodiments, the HRS polypeptidedoes not significantly compete for disease associated auto-antibodybinding to histidyl-tRNA synthetase in a competitive ELISA up to aconcentration of about 5×10⁻⁷M. In some embodiments, the HRS polypeptidedoes not significantly compete for disease associated auto-antibodybinding to histidyl-tRNA synthetase in a competitive ELISA up to aconcentration of about 1×10⁻⁶ M.

Accordingly in some embodiments, the HRS polypeptide has a loweraffinity to a disease associated auto-antibody than wild typehistidyl-tRNA synthetase (SEQ ID NO:1) as measured in a competitiveELISA. In some embodiments, the HRS polypeptide has an apparent affinityfor the disease associated auto-antibody which is at least about 10 foldless, or at least about 20 fold less, or at least about 50 fold less, orat least about 100 fold less than the affinity of the disease associatedauto-antibody to wild type human (SEQ ID NO:1). In one aspect, theauto-antibody to histidyl-tRNA synthetase is directed to the Jo-1antigen.

Examples of diseases associated with autoantibodies specific forhistidyl-tRNA synthetase (as well as diseases associated with aninsufficiency of histidyl-tRNA synthetase) include without limitation,autoimmune diseases, inflammatory diseases, and inflammatory myopathies,including idiopathic inflammatory myopathies, polymyositis, statininduced myopathies, dermatomyositis, interstitial lung disease (andother pulmonary fibrotic conditions) and related disorders, such aspolymyositis-scleroderma overlap and inclusion body myositis (IBM) andconditions such as those found in anti-synthetase syndromes, includingfor example, interstitial lung disease, arthritis, esophagealdysmotility, cardiovascular disease and other vascular manifestationssuch as Reynaud's phenomenon; other examples of diseases associated withan insufficiency of histidyl-tRNA synthetase include genetic disordersthat result in an insufficiency of active histidyl-tRNA synthetaseincluding Usher syndrome and Perrault syndrome.

Polymyositis affects skeletal muscles (involved with making movement) onboth sides of the body. It is rarely seen in persons under age 18; mostcases are in people between the ages of 31 and 60. In addition tosymptoms listed above, progressive muscle weakness leads to difficultyswallowing, speaking, rising from a sitting position, climbing stairs,lifting objects, or reaching overhead. People with polymyositis may alsoexperience arthritis, shortness of breath, and heart arrhythmias.Polymyositis is often associated with antibodies to synthetases,including HisRS, resulting in immune cell invasion into the damagedmuscle cells. HRS-Fc conjugates may thus be used to reduce immune cellactivation and invasion, and to treat polymyositis.

Dermatomyositis is characterized by a skin rash that precedes oraccompanies progressive muscle weakness. The rash looks patchy, withpurple or red discolorations, and characteristically develops on theeyelids and on muscles used to extend or straighten joints, includingknuckles, elbows, knees, and toes. Red rashes may also occur on theface, neck, shoulders, upper chest, back, and other locations, and theremay be swelling in the affected areas. The rash sometimes occurs withoutobvious muscle involvement. Adults with dermatomyositis may experienceweight loss or a low-grade fever, have inflamed lungs, and be sensitiveto light. Adult dermatomyositis, unlike polymyositis, may accompanytumors of the breast, lung, female genitalia, or bowel. Children andadults with dermatomyositis may develop calcium deposits, which appearas hard bumps under the skin or in the muscle (called calcinosis).Calcinosis most often occurs 1-3 years after disease onset but may occurmany years later. These deposits are seen more often in childhooddermatomyositis than in dermatomyositis that begins in adults.Dermatomyositis may be associated with collagen-vascular or autoimmunediseases.

In some cases of polymyositis and dermatomyositis, distal muscles (awayfrom the trunk of the body, such as those in the forearms and around theankles and wrists) may be affected as the disease progresses.Polymyositis and dermatomyositis may be associated withcollagen-vascular or autoimmune diseases resulting in immune cellinvasion into the damaged muscle cells. HRS-Fc conjugates may thus beused to reduce immune cell activation and invasion, and to treatdermatomyositis.

Inclusion body myositis (IBM) is characterized by progressive muscleweakness and wasting. The onset of muscle weakness is generally gradual(over months or years) and affects both proximal and distal muscles.Muscle weakness may affect only one side of the body. Small holes calledvacuoles are sometimes seen in the cells of affected muscle fibers.Falling and tripping are usually the first noticeable symptoms of IBM.For some individuals the disorder begins with weakness in the wrists andfingers that causes difficulty with pinching, buttoning, and grippingobjects. There may be weakness of the wrist and finger muscles andatrophy (thinning or loss of muscle bulk) of the forearm muscles andquadricep muscles in the legs. Difficulty swallowing occurs inapproximately half of IBM cases. Symptoms of the disease usually beginafter the age of 50, although the disease can occur earlier. Unlikepolymyositis and dermatomyositis, IBM occurs more frequently in men thanin women. As with other muscular dystrophies, IBM also results inprogressive immune cell invasion into the damaged muscle cells. HRS-Fcconjugates may thus be used to reduce immune cell activation andinvasion, and to treat IBM.

Juvenile myositis has some similarities to adult dermatomyositis andpolymyositis. It typically affects children ages 2 to 15 years, withsymptoms that include proximal muscle weakness and inflammation, edema(an abnormal collection of fluids within body tissues that causesswelling), muscle pain, fatigue, skin rashes, abdominal pain, fever, andcontractures (chronic shortening of muscles or tendons around joints,caused by inflammation in the muscle tendons, which prevents the jointsfrom moving freely). Children with juvenile myositis may also havedifficulty swallowing and breathing, and the heart may be affected.Approximately 20 to 30 percent of children with juvenile dermatomyositisdevelop calcinosis. Affected children may not show higher than normallevels of the muscle enzyme creatine kinase in their blood but havehigher than normal levels of other muscle enzymes. Juvenile myositisalso results in progressive immune cell invasion into the damaged musclecells. HRS-Fc conjugates may thus be used to reduce immune cellactivation and invasion, and to treat juvenile myositis.

Statin Induced Myopathies are associated with the long term use ofstatins which act via the inhibition of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR). Generally well-tolerated, thesemedications have been described as inducers of myotoxicity. Morerecently, there have been reports of patients in whom statin myopathiespersist even after drug cessation, which are hypothesized to have anautoimmune cause. The benefits of statins are undisputed in reducing therisk of coronary heart disease and the progression of coronaryatherosclerosis. Nevertheless, associated complications can belife-threatening. More than 38 million people in the U.S. are currentlyestimated to be taking statins and up to 7% (>2.6 million) of these arepredicted to develop muscle symptoms with up to 0.5% (>190,000) of thesepotentially going on to develop life-threatening myopathies.

All the statins can cause muscle problems and the risk increases alongwith increases in their lipophilicity, cholesterol-lowering potency, anddosage. Cerivastatin in particular has been implicated as having ahigher risk and it has been withdrawn from the US market. Of theremaining statins, atorvastatin and simvastatin have higher myotoxicityrates. Other nonstatin lipid-lowering agents such as niacin and fibratesalso carry risks of muscle problems, particularly when combined withstatins. While it is not possible to predict what patients will havestatin-induced muscle problems, prior muscle problems may be a riskfactor and should be considered when initiating statin treatment. Afamily history of myopathy is relevant if a patient might be a carrierof a genetic myopathy because it could be unmasked by the added stressof statin treatment. Other risk factors may include age over 80 years,low body weight, female sex, hypothyroidism, certain genetic defects andAsian descent, as well as concomitant use of certain medications,including calcium channel blockers, macrolide antibiotics, omeprazole,amiodarone, azole antifungals, histamine H2 receptor antagonists,nefazodone, cyclosporin, HIV protease inhibitors, warfarin, andgrapefruit juice.

The most common muscle symptom caused by statins is muscle pain ormyalgia and it occurs in about 7% of statin users. The myalgia can beanywhere from mild to severe and is often worsened by muscle activity.If the symptom is tolerable and the indication for statin treatmentstrong, for example, in a patient with hypercholesterolemia and a recentmyocardial infarction, continued statin treatment may be appropriate.

Baseline creatine kinase (CK) levels are not uniformly recommendedbefore initiation of statin treatment by the organizations guidingstatin treatment, but CK levels can provide very useful information ifmuscle symptoms later develop. Muscle weakness can also occur, and it isoften fatigable in quality and combined with pain and elevated CK. Likemost myopathies, the weakness is most pronounced proximally. Rareepisodes of rhabdomyolysis have also occurred with statin therapy; theseare far less frequent but can possibly be fatal. The changes that can beseen on muscle histology that are most typical of a statin myopathy arecytochrome oxidase negative fibers, increased lipid content, and raggedred fibers. Autoimmune necrotizing myopathy is a rare form of statinmyopathy. In these patients, discontinuation of the statin drug does nottranslate into recovery even after several months off the drug. Patientshave a predominantly proximal, often painless weakness.

Diagnosis is based on the individual's medical history, results of aphysical exam and tests of muscle strength, and blood samples that showelevated levels of various muscle enzymes and autoantibodies. Diagnostictools include electromyography to record the electrical activity thatcontrols muscles during contraction and at rest, ultrasound to look formuscle inflammation, and magnetic resonance imaging to reveal abnormalmuscle and evaluate muscle disease. A muscle biopsy can be examined bymicroscopy for signs of chronic inflammation, muscle fiber death,vascular deformities, or the changes specific to the diagnosis of IBM.HRS-Fc conjugates may thus be used to reduce immune cell activation andinvasion into damaged muscle, and to treat statin induced myopathies andrhabdomyolysis.

Interstitial lung disease (ILD) is a broad category of lung diseasesthat includes more than 130 disorders characterized by scarring (i.e.,“fibrosis”) and/or inflammation of the lungs. ILD accounts for 15percent of the cases seen by pulmonologists. Interstitial lung disease(ILD) can develop from a variety of sources, ranging from other diseasesto environmental factors. Some of the known causes of ILD include:connective tissue or autoimmune disease, including for example,scleroderma/progressive systemic sclerosis, lupus (systemic lupuserythematosus), rheumatoid arthritis and polymyositis/dermatomyositis;and occupational and environmental exposures, including for example,exposure to dust and certain gases, poisons, chemotherapy and radiationtherapy.

In ILD, the tissue in the lungs becomes inflamed and/or scarred. Theinterstitium of the lung includes the area in and around the small bloodvessels and alveoli (air sacs) where the exchange of oxygen and carbondioxide takes place. Inflammation and scarring of the interstitiumdisrupts this tissue and leads to a decrease in the ability of the lungsto extract oxygen from the air. HRS-Fc conjugates may thus be used toreduce immune cell activation and invasion into damaged lung, and totreat ILD.

The progression of ILD varies from disease to disease and from person toperson. Because interstitial lung disease disrupts the transfer ofoxygen and carbon dioxide in the lungs, its symptoms typically manifestas problems with breathing. The two most common symptoms of ILD areshortness of breath with exercise and a non-productive cough.

Usher Syndrome is the most common condition that affects both hearingand vision. The major symptoms of Usher syndrome are hearing loss andretinitis pigmentosa (RP). RP causes night-blindness and a loss ofperipheral vision (side vision) through the progressive degeneration ofthe retina. As RP progresses, the field of vision narrows until onlycentral vision remains. Many people with Usher syndrome also have severebalance problems. Approximately 3 to 6 percent of all children who aredeaf and another 3 to 6 percent of children who are hard-of-hearing haveUsher syndrome. In developed countries such as the United States, aboutfour babies in every 100,000 births have Usher syndrome. Usher syndromeis inherited as an autosomal recessive trait. Several genetic loci havebeen associated with Usher syndrome including histidyl t-RNA synthetase(Puffenberger et al., (2012) PLoS ONE 7(1) e28936 doi:10.1371/journal.pone.0028936).

There are three clinical types of Usher syndrome: type 1, type 2, andtype 3. In the United States, types 1 and 2 are the most common types.Together, they account for approximately 90 to 95 percent of all casesof children who have Usher syndrome.

Children with type 1 Usher syndrome are profoundly deaf at birth andhave severe balance problems. Because of the balance problems associatedwith type 1 Usher syndrome, children with this disorder are slow to sitwithout support and typically don't walk independently before they are18 months old. These children usually begin to develop vision problemsin early childhood, almost always by the time they reach age 10. Visionproblems most often begin with difficulty seeing at night, but tend toprogress rapidly until the person is completely blind.

Children with type 2 Usher syndrome are born with moderate to severehearing loss and normal balance. Although the severity of hearing lossvaries, most of these children can benefit from hearing aids and cancommunicate orally. The vision problems in type 2 Usher syndrome tend toprogress more slowly than those in type 1, with the onset of RP oftennot apparent until the teens.

Children with type 3 Usher syndrome have normal hearing at birth.Although most children with the disorder have normal to near-normalbalance, some may develop balance problems later on. Hearing and sightworsen over time, but the rate at which they decline can vary fromperson to person, even within the same family. A person with type 3Usher syndrome may develop hearing loss by the teens, and he or she willusually require hearing aids by mid- to late adulthood. Night blindnessusually begins sometime during puberty. Blind spots appear by the lateteens to early adulthood, and, by mid-adulthood, the person is usuallylegally blind.

Perrault syndrome (PS) is characterized by the association of ovariandysgenesis in females with sensorineural hearing impairment, and in somesubjects, neurologic abnormalities, including progressive cerebellarataxia and intellectual deficit. The exact prevalence for Perraultsyndrome is unknown, and is probably underdiagnosed, particularly inmales where hypogonadism is not a feature and the syndrome remainsundetected. Mean age at diagnosis is 22 years following presentationwith delayed puberty in females with sensorineural deafness. Hearingdefects were noted in all but one of the reported cases (mean age atdiagnosis of 8 years). The hearing loss is always sensorineural andbilateral but the severity is variable (mild to profound), even inaffected patients from the same family. Ovarian dysgenesis has beenreported in all female cases but no gonad defects are detected in males.Amenorrhea is generally primary but secondary amenorrhea has also beenreported. Delayed growth (height below the third percentile) wasreported in half the documented cases. The exact frequency of theneurological abnormalities is unknown, but nine females and two males(16-37 years old) lacking neurological abnormalities have been reported.Neurological signs are progressive and generally appear later in life,however, walking delay or early frequent falls have been noted in youngPS patients. Common neurological signs are ataxia, dyspraxia, limitedextraocular movements, and polyneuropathy. Some cases with scoliosishave also been reported. Transmission of PS is autosomal recessive andmutations in mitochondrial histidyl tRNA synthetase have recently beenidentified to cause the ovarian dysgenesis and sensorineural hearingloss associated with Perrault syndrome. (Pierce et al., PNAS USA.108(16) 6543-6548, 2011).

Muscular dystrophy refers to a group of inherited disorders in whichstrength and muscle bulk gradually decline. All of the musculardystrophies are marked by muscle weakness that is driven by a primarygenetic defect in one or more muscle specific genes. Additionallymuscular dystrophies, typically have a variable inflammatory componentthat drives muscular inflammation and ultimately enhances thedegeneration of muscular tissues. Accordingly HRS-Fc conjugates may beused to reduce immune cell activation and invasion into damaged muscle,and to treat muscular dystrophies. At least nine types of musculardystrophies are generally recognized. In some aspects, the musculardystrophy is selected from Duchenne muscular dystrophy, Becker musculardystrophy, Emery-Dreifuss muscular dystrophy, Limb-girdle musculardystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy,oculopharyngeal muscular dystrophy, distal muscular dystrophy andcongenital muscular dystrophy.

Duchenne muscular dystrophy (DMD): DMD affects young boys, causingprogressive muscle weakness, usually beginning in the legs. It is themost severe form of muscular dystrophy. DMD occurs in about 1 in 3,500male births, and affects approximately 8,000 boys and young men in theUnited States. A milder form occurs in very few female carriers.

DMD is caused by mutations in the gene encoding dystrophin, asubsarcolemmal protein functioning within the dystrophin-associatedglycoprotein complex (DGC) which prevent the production of functionalprotein. The amount of dystrophin correlates with the severity of thedisease (i.e., the less dystrophin present, the more severe thephenotype). The DGC complex connects the intracellular cytoskeleton tothe extracellular matrix. The DGC is concentrated at the Z-lines of thesarcomere and confers the transmission of force across the muscle fibre.Disruption of this link results in membrane instability, whicheventually leads to sarcolemmal ruptures. Influx of extracellularcalcium alters molecular processes like muscle contraction and activatesproteolytic activity. Affected muscle fibres become necrotic orapoptotic, and release mitogenic chemoattractants, which initiateinflammatory processes. Cycles of degeneration and regenerationeventually lead to irreversible muscle wasting and replacement byfibrotic and adipose tissue.

A boy with Duchenne muscular dystrophy usually begins to show symptomsas a pre-schooler. The legs are affected first, making walking difficultand causing balance problems. Most patients walk three to six monthslater than expected and have difficulty running Contractures (permanentmuscle tightening) usually begin by age five or six, most severely inthe calf muscles. Frequent falls and broken bones are common beginningat this age. Climbing stairs and rising unaided may become impossible byage nine or ten, and most boys use a wheelchair for mobility by the ageof 12. Weakening of the trunk muscles around this age often leads toscoliosis (a side-to-side spine curvature) and kyphosis (a front-to backcurvature).

One of the most serious weakness of DMD is weakness of the diaphragm,the sheet of muscles at the top of the abdomen that perform the mainwork of breathing and coughing. Diaphragm weakness leads to reducedenergy and stamina, and increased lung infection because of theinability to cough effectively. Young men with DMD can live into theirtwenties and beyond, provided they have mechanical ventilationassistance and good respiratory hygiene.

In some embodiments, a subject having DMD is characterized by one ormore of the following: a positive Gower's sign, reflecting impairment ofthe lower extremity muscles; high levels of creatine kinase (CPK-MM) inthe blood; genetic errors in the Xp21 gene; or reduced levels of absenceof dystrophin, for instance, as measured by muscle biopsy.

HRS-Fc conjugates may be used in the treatment of DMD, either alone orin combination with other therapies, such as antisense oligonucleotides(e.g., exon-skipping therapies such as Eteplirsen), corticosteroids,beta2-agonists, physical therapy, respiratory support, stem celltherapies, and gene replacement therapies. In some embodiments,administration of HRS-Fc conjugates leads to statistically significantimprovements in the 6-minute walk test.

Becker muscular dystrophy (BMD): BMD affects older boys and young men,following a milder course than DMD. BMD occurs in about 1 in 30,000 malebirths. Becker muscular dystrophy is a less severe variant of Duchennemuscular dystrophy and is caused by the production of a truncated, butpartially functional form of dystrophin.

The symptoms of BMD usually appear in late childhood to early adulthood.Though the progression of symptoms may parallel that of DMD, thesymptoms are usually milder and the course more variable. Scoliosis mayoccur, but is usually milder and progresses more slowly. Heart muscledisease (cardiomyopathy), occurs more commonly in BMD. Problems mayinclude irregular heartbeats (arrhythmias) and congestive heart failure.Symptoms may include fatigue, shortness of breath, chest pain, anddizziness. Respiratory weakness also occurs, and may lead to the needfor mechanical ventilation. HRS-Fc conjugates may be used in thetreatment of BMD, either alone or in combination with other therapies.

Emery-Dreifuss muscular dystrophy (EDMD): EDMD affects young boys,causing contractures and weakness in the calves, weakness in theshoulders and upper arms, and problems in the way electrical impulsestravel through the heart to make it beat (heart conduction defects).There are three subtypes of Emery-Dreifuss Muscular Dystrophy,distinguishable by their pattern of inheritance: X-Linked, autosomaldominant and autosomal recessive. The X-linked form is the most common.Each type varies in prevalence and symptoms. The disease is caused bymutations in the LMNA gene, or more commonly, the EMD gene. Both genesencode for protein components of the nuclear envelope.

EDMD usually begins in early childhood, often with contracturespreceding muscle weakness. Weakness affects the shoulder and upper armoriginally, along with the calf muscles, leading to foot-drop. Most menwith EDMD survive into middle age, although a defect in the heart'srhythm (heart block) may be fatal if not treated with a pacemaker.HRS-Fc conjugates may be used in the treatment of EDMD, either alone orin combination with other therapies.

Limb-girdle muscular dystrophy (LGMD): LGMD begins in late childhood toearly adulthood and affects both men and women, causing weakness in themuscles around the hips and shoulders. It is the most variable of themuscular dystrophies, and there are several different forms of thedisease now recognized. Many people with suspected LGMD have probablybeen misdiagnosed in the past, and therefore the prevalence of thedisease is difficult to estimate. The number of people affected in theUnited States may be in the low thousands.

While there are at least a half-dozen genes that cause the various typesof LGMD, two major clinical forms of LGMD are usually recognized. Asevere childhood form is similar in appearance to DMD, but is inheritedas an autosomal recessive trait.

Limb Girdle Muscular Dystrophy type 2B (LGMD2B) is caused by the loss offunction mutations in the dysferlin gene. Dysferlin is primarilyexpressed in skeletal and cardiac muscle, but also in monocytes,macrophages, and other tissues where it is localized to cytoplasmicvesicles and the cell membrane. Dysferlin appears to be involved inmembrane fusion and trafficking, as well as repair processes. LGMD2B isa late onset (teens/young adults) muscle disease that is characterizedby progressive symmetrical muscle weakness, and notably aggressiveimmune/inflammatory pathology. Muscle biopsies typically show markedinflammatory cell infiltration, consisting primarily ofmacrophages/macrophage activation markers (HLA-DR, HLA-ABC, CD86), CD8⁺cytotoxic T cells, and CD4⁺ T cells, together with muscle fiberdegeneration/regeneration. Accordingly, HRS-Fc conjugates may be used toreduce immune cell activation and invasion into damaged muscle, and totreat Limb Girdle Muscular Dystrophy.

Symptoms of adult-onset LGMD usually appear in a person's teens ortwenties, and are marked by progressive weakness and wasting of themuscles closest to the trunk. Contractures may occur, and the ability towalk is usually lost about 20 years after onset. Some people with LGMDdevelop respiratory weakness that requires use of a ventilator. Lifespanmay be somewhat shortened. (Autosomal dominant forms usually occur laterin life and progress relatively slowly.)

Facioscapulohumeral muscular dystrophy (FSH): FSH, also known asLandouzy-Dejerine disease, begins in late childhood to early adulthoodand affects both men and women, causing weakness in the muscles of theface, shoulders, and upper arms. The hips and legs may also be affected.FSH occurs in about 1 out of every 20,000 people, and affectsapproximately 13,000 people in the United States.

FSH varies in its severity and age of onset, even among members of thesame family. Symptoms most commonly begin in the teens or earlytwenties, though infant or childhood onset is possible. Symptoms tend tobe more severe in those with earlier onset. The disease is named for theregions of the body most severely affected by the disease: muscles ofthe face (facio-), shoulders (scapulo-), and upper arms (humeral). Hipsand legs may be affected as well. Children with FSH often developpartial or complete deafness.

Two defects are needed for FSHD, the first is the deletion of D4Z4repeats and the second is a “toxic gain of function” of the DUX4 gene.The first symptom noticed is often difficulty lifting objects above theshoulders. The weakness may be greater on one side than the other.Shoulder weakness also causes the shoulder blades to jut backward,called scapular winging. FSHD is associated with inflammatory invasionis specific muscle groups, and accordingly HRS-Fc conjugates may thus beused to reduce immune cell activation and invasion into damaged muscles,and to treat FSHD.

Myotonic dystrophy: Myotonic dystrophy, also known as Steinert'sdisease, affects both men and women, causing generalized weakness firstseen in the face, feet, and hands. It is accompanied by the inability torelax the affected muscles (myotonia). Symptoms may begin from birththrough adulthood. Myotonic muscular dystrophy type 1 (DM1) is the mostcommon form of muscular dystrophy, affecting more than 30,000 people inthe United States. It results from the expansion of a short (CTG) repeatin the DNA sequence of the DMPK (myotonic dystrophy protein kinase)gene. Myotonic muscular dystrophy type 2 (DM2) is much rarer and is aresult of the expansion of the CCTG repeat in the ZNF9 (zinc fingerprotein 9) gene.

Symptoms of myotonic dystrophy include facial weakness and a slack jaw,drooping eyelids (ptosis), and muscle wasting in the forearms andcalves. A person with this dystrophy has difficulty relaxing his grasp,especially if the object is cold. Myotonic dystrophy affects heartmuscle, causing arrhythmias and heart block, and the muscles of thedigestive system, leading to motility disorders and constipation. Otherbody systems are affected as well: Myotonic dystrophy may causecataracts, retinal degeneration, low IQ, frontal balding, skindisorders, testicular atrophy, sleep apnea, and insulin resistance. Anincreased need or desire for sleep is common, as is diminishedmotivation. Severe disability affects most people with this type ofdystrophy within 20 years of onset, although most do not require awheelchair even late in life. HRS-Fc conjugates can thus be used totreat myotonic dystrophy, for instance, by reducing inflammationassociated with muscle tissue, including skeletal muscle (e.g.,quadricep muscles) and/or heart tissue, among other tissues.

Oculopharyngeal muscular dystrophy (OPMD): OPMD affects adults of bothsexes, causing weakness in the eye muscles and throat. It is most commonamong French Canadian families in Quebec, and in Spanish-Americanfamilies in the southwestern United States.

OPMD usually begins in a person's thirties or forties, with weakness inthe muscles controlling the eyes and throat. Symptoms include droopingeyelids, difficulty swallowing (dysphagia), and weakness progresses toother muscles of the face, neck, and occasionally the upper limbs.Swallowing difficulty may cause aspiration, or the introduction of foodor saliva into the airways. Pneumonia may follow. HRS-Fc conjugates canthus be used to treat OPMD, for instance, by reducing inflammationassociated with muscle tissue.

Distal muscular dystrophy (DD): DD begins in middle age or later,causing weakness in the muscles of the feet and hands. It is most commonin Sweden, and rare in other parts of the world. DD usually begins inthe twenties or thirties, with weakness in the hands, forearms, andlower legs. Difficulty with fine movements such as typing or fasteningbuttons may be the first symptoms. Symptoms progress slowly, and thedisease usually does not affect life span. HRS-Fc conjugates can thus beused to treat DD, by reducing inflammation associated with muscle tissueinflammation.

Congenital muscular dystrophy (CMD): CMD is present from birth, resultsin generalized weakness, and usually progresses slowly. A subtype,called Fukuyama CMD, also involves mental retardation. Both are rare;Fukuyama CMD is more common in Japan.

CMD is marked by severe muscle weakness from birth, with infantsdisplaying “floppiness” and very little voluntary movement. Nonetheless,a child with CMD may learn to walk, either with or without someassistive device, and live into young adulthood or beyond. In contrast,children with Fukuyama CMD are rarely able to walk, and have severemental retardation. Most children with this type of CMD die inchildhood. As with the other muscular dystrophies, HRS-Fc conjugates canthus be used to treat CMD, for example, by reducing inflammationassociated with muscle tissue inflammation.

Cachexia: Cachexia (or wasting syndrome) is typically characterized byloss of weight, muscle atrophy, fatigue, weakness, and significant lossof appetite in someone who is not actively trying to lose weight. Theformal definition of cachexia is the loss of body mass that cannot bereversed nutritionally. Even if the affected patient consumes morecalories, lean body mass is lost, indicating the existence of a primarypathology.

Cachexia is experienced by patients with cancer, AIDS, chronicobstructive lung disease, multiple sclerosis, congestive heart failure,tuberculosis, familial amyloid polyneuropathy, mercury poisoning(acrodynia), and hormonal deficiency, among other disease.

Cachexia can also be a sign of various underlying disorders, includingcancer, metabolic acidosis (i.e., decreased protein synthesis andincreased protein catabolism), certain infectious diseases (e.g.,tuberculosis, AIDS), chronic pancreatitis, autoimmune disorders, oraddiction to amphetamines. Cachexia physically weakens patients to astate of immobility stemming from loss of appetite, asthenia, andanemia, and response to standard treatment is usually poor.

About 50% of all cancer patients suffer from cachexia. Those with uppergastrointestinal and pancreatic cancers have the highest frequency ofdeveloping a cachexic symptom. In addition to increasing morbidity andmortality, aggravating the side effects of chemotherapy, and reducingquality of life, cachexia is considered the immediate cause of death ofa large proportion of cancer patients, ranging from 22% to 40% of thepatients. Symptoms of cancer cachexia include progressive weight lossand depletion of host reserves of adipose tissue and skeletal muscle.Traditional treatment approaches include the use of appetite stimulants,5-HT₃ antagonists, nutrient supplementation, and COX-2 inhibitors.

Although the pathogenesis of cachexia is poorly understood, multiplebiologic pathways are expected to be involved, includingpro-inflammatory cytokines such as TNF-α, neuroendocrine hormones,IGF-1, and tumor-specific factors such as proteolysis-inducing factor.

HRS-Fc conjugates may thus be used to treat cachexia and any of itsrelated, underlying, or secondary disorders or complications. HRS-Fcconjugates can be used alone or in combination with other therapies,such as dietary supplementation with a combination of high protein,leucine and fish oil, antioxidants, progestogen (megestrol acetate,medroxyprogesterone acetate), and anticyclooxygenase-2 drugs, appetitestimulants, and 5-HT3 antagonists, among others.

Rhabdomyolysis: Rhabdomyolysis is the breakdown of muscle fibers inskeletal muscle tissue. The breakdown products are released into thebloodstream, and certain some of these products, such as myoglobin, areharmful to the kidneys and may lead to kidney failure.

Symptoms include muscle pain, vomiting, confusion, coma, or abnormalheart rate and rhythm and their severity usually depends on the extentof muscle damage and whether kidney failure develops. Damage to thekidneys may cause decreased or absent urine production, usually about 12to 24 hours after the initial muscle damage. Swelling of the damagedmuscle can cause compartment syndrome, or compression of surroundingtissues, such as nerves and blood vessels, in the same fascialcompartment, and lead to blood loss and damage to (e.g., loss offunction) the affected body parts. Symptoms of this complication includepain or reduced sensation in the affected limb. Other complicationsinclude disseminated intravascular coagulation (DIC), a severedisruption in blood clotting that may lead to uncontrollable bleeding

The initial muscle damage may be caused, for instance, by physicalfactors (e.g. crush injury, strenuous exercise), altered blood supply(e.g., arterial thrombosis, embolism), altered metabolism (e.g.,hyperglycemic hyperosmolar state, hyper- and hyponatremia, hypokalemia,hypocalcemia, hypophosphatemia, ketoacidosis, hypothyroidism), alteredbody temperature (hyperthermia, hypothermia), medications and toxins(e.g., statins, anti-psychotic medications, neuromuscular blockingagents, diuretics, heavy metals, hemlock, insect or snake venoms), drugabuse (e.g., alcohol, amphetamine, cocaine, heroin, ketamine, LDS,MDMA), infections (e.g., Coxsackie virus, influenza A virus, influenza Bvirus, Epstein-Barr virus, primary HIV infection, Plasmodium falciparum,herpes viruses, Legionella pneumophila, salmonella), and autoimmunemuscle damage (e.g., polymyositis, dermatomyositis). Also, certainhereditary conditions increase the risk of rhabdomyolysis, includingglycolysis and glycogenolysis defects (e.g., McArdle's disease,phosphofructokinase deficiency, glycogen storage diseases VIII, IX, Xand XI), lipid metabolism defects (e.g., carnitine palmitoyltransferaseI and II deficiency, deficiency of subtypes of acyl CoA dehydrogenase(e.g., LCAD, SCAD, MCAD, VLCAD, 3-hydroxyacyl-coenzyme A dehydrogenasedeficiency), thiolase deficiency), mitochondrial myopathies (e.g.,deficiency of succinate dehydrogenase, cytochrome c oxidase and coenzymeQ10), and others such as glucose-6-phosphate dehydrogenase deficiency,myoadenylate deaminase deficiency, and muscular dystrophies.

Rhabdomyolysis is usually diagnosed with blood tests and urinalysis, andcan be indicated by abnormally raised or increasing creatinine and urealevels, falling urine output, or reddish-brown discoloration of theurine. The primary treatments include intravenous fluids, dialysis, andhemofiltration.

HRS-Fc conjugates may thus be used to treat rhabdomyolysis and any ofits related, secondary, or underlying disorders or complications. HRS-Fcconjugates can be used alone or in combination with other therapies,including those meant to treat shock and preserve kidney function.Exemplary therapies include administration of intravenous fluids,usually isotonic saline (0.9% weight per volume sodium chloridesolution) and renal replacement therapies (RRT) such as hemodialysis,continuous hemofiltration and peritoneal dialysis.

More generally, the HRS-Fc conjugates described herein can reduce aninflammatory response, such as by reducing the activation,differentiation, migration, or infiltration of immune cells intoselected tissues, increasing the production of anti-inflammatorycytokines, or reducing the production or activity of pro-inflammatorycytokines, among other mechanisms. Moreover, certain of the presentmethods, by blocking the binding, action, or production ofanti-histidyl-tRNA synthetase antibodies or auto-reactive T cells, haveutility to treat a broad range of auto-immune and inflammatory diseasesand disorders associated with anti-histidyl-tRNA synthetase antibodies,other auto-antibodies, as well as other causes of histidyl-tRNAsynthetase insufficiency.

Pharmaceutical Formulations, Administration, and Kits

Embodiments of the present invention include compositions comprisingHRS-Fc conjugate polypeptides formulated in pharmaceutically-acceptableor physiologically-acceptable solutions for administration to a cell,subject, or an animal, either alone, or in combination with one or moreother modalities of therapy. It will also be understood that, ifdesired, the compositions of the invention may be administered incombination with other agents as well, for example, other proteins orpolypeptides or various pharmaceutically-active agents. There isvirtually no limit to other components that may also be included in thecompositions, provided that the additional agents do not adverselyaffect the modulatory or other effects desired to be achieved.

For pharmaceutical production, HRS-Fc conjugate therapeutic compositionswill typically be substantially endotoxin free. Endotoxins are toxinsassociated with certain bacteria, typically gram-negative bacteria,although endotoxins may be found in gram-positive bacteria, such asListeria monocytogenes. The most prevalent endotoxins arelipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in theouter membrane of various Gram-negative bacteria, and which represent acentral pathogenic feature in the ability of these bacteria to causedisease. Small amounts of endotoxin in humans may produce fever, alowering of the blood pressure, and activation of inflammation andcoagulation, among other adverse physiological effects.

Endotoxins can be detected using routine techniques known in the art.For example, the Limulus Amoebocyte Lysate assay, which utilizes bloodfrom the horseshoe crab, is a very sensitive assay for detectingpresence of endotoxin. In this test, very low levels of LPS can causedetectable coagulation of the limulus lysate due a powerful enzymaticcascade that amplifies this reaction. Endotoxins can also be quantitatedby enzyme-linked immunosorbent assay (ELISA).

To be substantially endotoxin free, endotoxin levels may be less thanabout 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1,0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of protein.Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

In certain embodiments, as noted herein, the HRS-Fc conjugatecompositions have an endotoxin content of less than about 10 EU/mg ofHRS-Fc conjugate, or less than about 5 EU/mg of HRS-Fc conjugate, lessthan about 3 EU/mg of HRS-Fc conjugate, or less than about 1 EU/mg ofHRS-Fc conjugate or less than about 0.1 EU/mg of HRS-Fc conjugate, orless than about 0.01EU/mg of HRS-Fc conjugate. In certain embodiments,as noted above, the HRS-Fc conjugate pharmaceutical compositions areabout 95% endotoxin free, preferably about 99% endotoxin free, and morepreferably about 99.99% endotoxin free on wt/wt protein basis.

Pharmaceutical compositions comprising a therapeutic dose of a HRS-Fcconjugate polypeptide include all homologues, orthologs, andnaturally-occurring isoforms of histidyl-tRNA synthetase.

In some embodiments such pharmaceutical compositions may comprise ahistidine buffer, which may be present in any of the pharmaceuticalcompositions within the range of about 1 mM to about 100 mM. In someembodiments, the histidine buffer may be present at a concentration ofabout 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM,about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM,about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM,or about 100 mM, including all integers and ranges in between saidconcentrations.

In one aspect such compositions may comprises HRS-Fc conjugatepolypeptides that are substantially monodisperse, meaning that theHRS-Fc conjugate compositions exist primarily (i.e., at least about 90%,or greater) in one apparent molecular weight form when assessed forexample, by size exclusion chromatography, dynamic light scattering, oranalytical ultracentrifugation.

In another aspect, such compositions have a purity (on a protein basis)of at least about 90%, or in some aspects at least about 95% purity, orin some embodiments, at least 98% purity. Purity may be determined viaany routine analytical method as known in the art.

In another aspect, such compositions have a high molecular weightaggregate content of less than about 10%, compared to the total amountof protein present, or in some embodiments such compositions have a highmolecular weight aggregate content of less than about 5%, or in someaspects such compositions have a high molecular weight aggregate contentof less than about 3%, or in some embodiments a high molecular weightaggregate content of less than about 1%. High molecular weight aggregatecontent may be determined via a variety of analytical techniquesincluding for example, by size exclusion chromatography, dynamic lightscattering, or analytical ultracentrifugation.

Pharmaceutical compositions may include pharmaceutically acceptablesalts of a HRS-Fc conjugate polypeptide. For a review on suitable salts,see Handbook of Pharmaceutical Salts: Properties, Selection, and Use byStahl and Wermuth (Wiley-VCH, 2002). Suitable base salts are formed frombases which form non-toxic salts. Representative examples include thealuminum, arginine, benzathine, calcium, choline, diethylamine,diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium,sodium, tromethamine, and zinc salts. Hemisalts of acids and bases mayalso be formed, e.g., hemisulphate and hemicalcium salts. Compositionsto be used in the invention suitable for parenteral administration maycomprise sterile aqueous solutions and/or suspensions of thepharmaceutically active ingredients preferably made isotonic with theblood of the recipient, generally using sodium chloride, glycerin,glucose, mannitol, sorbitol, and the like. Organic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, acetic acid, trifluoroacetic acid,propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolicacid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamicacid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid), arylsulfonic acids (e.g., benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid),4-methylbicyclo(2.2.2)-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like.

In particular embodiments, the carrier may include water. In someembodiments, the carrier may be an aqueous solution of saline, forexample, water containing physiological concentrations of sodium,potassium, calcium, magnesium, and chloride at a physiological pH. Insome embodiments, the carrier may be water and the formulation mayfurther include NaCl. In some embodiments, the formulation may beisotonic. In some embodiments, the formulation may be hypotonic. Inother embodiments, the formulation may be hypertonic. In someembodiments, the formulation may be isomostic. In some embodiments, theformulation is substantially free of polymers (e.g., gel-formingpolymers, polymeric viscosity-enhancing agents). In some embodiments,the formulation is substantially free of viscosity-increasing agents(e.g., carboxymethylcellulose, polyanionic polymers). In someembodiments, the formulation is substantially free of gel-formingpolymers. In some embodiments, the viscosity of the formulation is aboutthe same as the viscosity of a saline solution containing the sameconcentration of a HRS-Fc conjugate (or a pharmaceutically acceptablesalt thereof).

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

In certain embodiments, the HRS-Fc conjugate polypeptide have asolubility that is desirable for the particular mode of administration,such intravenous administration. Examples of desirable solubility'sinclude at least about 1 mg/ml, at least about 10 mg/ml, at least about25 mg/ml, and at least about 50 mg/ml.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anedible carrier, or they may be enclosed in hard- or soft-shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet.

Pharmaceutical compositions suitable for the delivery of HRS-Fcconjugates and methods for their preparation will be readily apparent tothose skilled in the art. Such compositions and methods for theirpreparation may be found, for example, in Remington's PharmaceuticalSciences, 19th Edition (Mack Publishing Company, 1995).

Administration of a therapeutic dose of a HRS-Fc conjugate may be by anysuitable method known in the medicinal arts, including for example,oral, intranasal, parenteral administration include intravitreal,subconjuctival, sub-tenon, retrobulbar, suprachoroidal intravenous,intra-arterial, intraperitoneal, intrathecal, intraventricular,intraurethral, intrasternal, intracranial, intramuscular, intrasynovial,intraocular, topical and subcutaneous. Suitable devices for parenteraladministration include needle (including microneedle) injectors,needle-free injectors, and infusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates, and buffering agents(preferably to a pH of from 3 to 9), but, for some applications, theymay be more suitably formulated as a sterile non-aqueous solution or asa dried form to be used in conjunction with a suitable vehicle such assterile, pyrogen-free water. The preparation of parenteral formulationsunder sterile conditions, for instance, by lyophilization, may readilybe accomplished using standard pharmaceutical techniques well-known tothose skilled in the art.

Formulations for parenteral administration may be formulated to beimmediate and/or sustained release. Sustained release compositionsinclude delayed, modified, pulsed, controlled, targeted and programmedrelease. Thus a HRS-Fc conjugate may be formulated as a suspension or asa solid, semi-solid, or thixotropic liquid for administration as animplanted depot providing sustained release of HRS-Fc conjugates.Examples of such formulations include without limitation, drug-coatedstents and semi-solids and suspensions comprising drug-loadedpoly(DL-lactic-co-glycolic)acid (PGLA), poly(DL-lactide-co-glycolide)(PLG) or poly(lactide) (PLA) lamellar vesicles or microparticles,hydrogels (Hoffman AS: Ann. N.Y. Acad. Sci. 944: 62-73 (2001)),poly-amino acid nanoparticles systems, such as the Medusa systemdeveloped by Flamel Technologies Inc., non aqueous gel systems such asAtrigel developed by Atrix, Inc., and SABER (Sucrose Acetate IsobutyrateExtended Release) developed by Durect Corporation, and lipid-basedsystems such as DepoFoam developed by SkyePharma.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, incorporated by reference in itsentirety). In all cases the form should be sterile and should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol,liquid polyethylene glycol, and the like), suitable mixtures thereof,and/or vegetable oils. Proper fluidity may be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can befacilitated by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

HRS-Fc conjugate polypeptides for use in the present invention may alsobe administered topically, (intra)dermally, or transdermally to theskin, mucosa, or surface of the eye, either alone or in combination withone or more antihistamines, one or more antibiotics, one or moreantifungal agents, one or more beta blockers, one or moreanti-inflammatory agents, one or more antineoplastic agents, one or moreimmunosuppressive agents, one or more antiviral agents, one or moreantioxidant agents, or other active agents. Formulations for topical andocular administration may be formulated to be immediate and/or modifiedrelease. Modified release formulations include delayed, sustained,pulsed, controlled, targeted and programmed release.

Typical formulations for this purpose include gels, hydrogels, lotions,solutions, eye drops, creams, ointments, dusting powders, dressings,foams, films, skin patches, wafers, implants, sponges, fibers, bandages,and microemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol, and propylene glycol. Penetrationenhancers may be incorporated—see, e.g., Finnin and Morgan: J. Pharm.Sci. 88(10): 955-958, (1999). Other means of topical administrationinclude delivery by electroporation, iontophoresis, phonophoresis,sonophoresis, and microneedle or needle-free injection (e.g., thesystems sold under the trademarks POWDERJECT™, BIOJECT™).

Examples of antihistamines include, but are not limited to, loradatine,hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine,cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine,diphenylpyraline, phenindamine, azatadine, tripelennamine,dexchlorpheniramine, dexbrompheniramine, methdilazine, and trimprazinedoxylamine, pheniramine, pyrilamine, chiorcyclizine, thonzylamine, andderivatives thereof.

Examples of antibiotics include, but are not limited to, aminoglycosides(e.g., amikacin, apramycin, arbekacin, bambermycins, butirosin,dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin,kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin,tobramycin, trospectomycin), amphenicols (e.g., azidamfenicol,chloramphenicol, florfenicol, thiamphenicol), ansamycins (e.g.,rifamide, rifampin, rifamycin sv, rifapentine, rifaximin), lactams(e.g., carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem,imipenem, meropenem, panipenem), cephalosporins (e.g., cefaclor,cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefcapenepivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime,cefinenoxime, cefodizime, cefonicid, cefoperazone, ceforanide,cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome,cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime,cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin,cephaloridine, cephalosporin, cephalothin, cephapirin sodium,cephradine, pivcefalexin), cephamycins (e.g., cefbuperazone,cefinetazole, cefininox, cefotetan, cefoxitin), monobactams (e.g.,aztreonam, carumonam, tigemonam), oxacephems, flomoxef, moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,carbenicillin, carindacillin, clometocillin, cloxacillin, cyclacillin,dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin,lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillinsodium, oxacillin, penamecillin, penethamate hydriodide, penicillin gbenethamine, penicillin g benzathine, penicillin g benzhydrylamine,penicillin g calcium, penicillin g hydrabamine, penicillin g potassium,penicillin g procaine, penicillin n, penicillin o, penicillin v,penicillin v benzathine, penicillin v hydrabamine, penimepicycline,phenethicillin potassium, piperacillin, pivampicillin, propicillin,quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin,ticarcillin), other (e.g., ritipenem), lincosamides (e.g., clindamycin,lincomycin), macrolides (e.g., azithromycin, carbomycin, clarithromycin,dirithromycin, erythromycin, erythromycin acistrate, erythromycinestolate, erythromycin glucoheptonate, erythromycin lactobionate,erythromycin propionate, erythromycin stearate, josamycin, leucomycins,midecamycins, miokamycin, oleandomycin, primycin, rokitamycin,rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides(e.g., amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, fusafungine, gramicidin s, gramicidin(s), mikamycin,polymyxin, pristinamycin, ristocetin, teicoplanin, thiostrepton,tuberactinomycin, tyrocidine, tyrothricin, vancomycin, viomycin,virginiamycin, zinc bacitracin), tetracyclines (e.g., apicycline,chlortetracycline, clomocycline, demeclocycline, doxycycline,guamecycline, lymecycline, meclocycline, methacycline, minocycline,oxytetracycline, penimepicycline, pipacycline, rolitetracycline,sancycline, tetracycline), and others (e.g., cycloserine, mupirocin,tuberin). 2,4-Diaminopyrimidines (e.g., brodimoprim, tetroxoprim,trimethoprim), nitrofurans (e.g., furaltadone, furazolium chloride,nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine,nifurtoinol, nitrofurantoin), quinolones and analogs (e.g., cinoxacin,ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin,flumequine, grepafloxacin, lomefloxacin, miloxacin, nadifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pazufloxacin,pefloxacin, pipemidic acid, piromidic acid, rosoxacin, rufloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin), sulfonamides(e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, chloramine-b,chloramine-t, dichloramine t, n²-formylsulfisomidine, mafenide,4′-(methylsulfamoyl)sulfanilanilide, noprylsulfamide,phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine,succinylsulfathiazole, sulfabenzamide, sulfacetamide,sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine,sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole,sulfaguanidine, sulfaguanol, sulfalene, sulfaloxic acid, sulfamerazine,sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine,sulfamethoxazole, sulfamethoxypyridazine, sulfametrole,sulfamidochrysoidine, sulfamoxole, sulfanilamide,4-sulfanilamidosalicylic acid, n⁴-sulfanilylsulfanilamide,sulfanilylurea, n-sulfanilyl-3,4-xylamide, sulfanitran, sulfaperine,sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine,sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea,sulfatolamide, sulfisomidine, sulfisoxazole) sulfones (e.g., acedapsone,acediasulfone, acetosulfone sodium, dapsone, diathymosulfone,glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid,p-sulfanilylbenzylamine, sulfoxone sodium, thiazolsulfone), and others(e.g., clofoctol, hexedine, methenamine, methenamineanhydromethylene-citrate, methenamine hippurate, methenamine mandelate,methenamine sulfosalicylate, nitroxoline, taurolidine, xibornol).

Examples of antifungal agents include, but are not limited to Polyenes(e.g., amphotericin b, candicidin, dermostatin, filipin, fungichromin,hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin,pecilocin, perimycin), others (e.g., azaserine, griseofulvin,oligomycins, neomycin undecylenate, pyrrolnitrin, siccanin, tubercidin,viridin), Allylamines (e.g., butenafine, naftifine, terbinafine),imidazoles (e.g., bifonazole, butoconazole, chlordantoin, chlormidazole,cloconazole, clotrimazole, econazole, enilconazole, fenticonazole,flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole,omoconazole, oxiconazole nitrate, sertaconazole, sulconazole,tioconazole), thiocarbamates (e.g., tolciclate, tolindate, tolnaftate),triazoles (e.g., fluconazole, itraconazole, saperconazole, terconazole)others (e.g., acrisorcin, amorolfine, biphenamine,bromosalicylchloranilide, buclosamide, calcium propionate,chlorphenesin, ciclopirox, cloxyquin, coparaffinate, diamthazoledihydrochloride, exalamide, flucytosine, halethazole, hexetidine,loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione,salicylanilide, sodium propionate, sulbentine, tenonitrozole, triacetin,ujothion, undecylenic acid, zinc propionate).

Examples of beta blockers include but are not limited to acebutolol,atenolol, labetalol, metoprolol, propranolol, timolol, and derivativesthereof.

Examples of antineoplastic agents include, but are not limited toantibiotics and analogs (e.g., aclacinomycins, actinomycin f₁,anthramycin, azaserine, bleomycins, cactinomycin, carubicin,carzinophilin, chromomycins, dactinomycin, daunorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin,menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines,peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin,streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin),antimetabolites (e.g. folic acid analogs (e.g., denopterin, edatrexate,methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purineanalogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine,thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, doxifluridine, emitefur,enocitabine, floxuridine, fluorouracil, gemcitabine, tagafur).

Examples of anti-inflammatory agents include but are not limited tosteroidal anti-inflammatory agents and non-steroidal anti-inflammatoryagents. Exemplary steroidal anti-inflammatory agents includeacetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, and triamcinolonehexacetonide.

Exemplary non-steroidal anti-inflammatory agents includeaminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate,flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumicacid, talniflumate, terofenamate, tolfenamic acid), arylacetic acidderivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac,amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac,diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin,sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acidderivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin),arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine),arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen,bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen,flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen,naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinicacid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles(e.g., difenamizole, epirizole), pyrazolones (e.g., apazone,benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone,phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone,thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol,aspirin, benorylate, bromosaligenin, calcium acetylsalicylate,diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate,imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholinesalicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenylacetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-aceticacid, salicylsulfuric acid, salsalate, sulfasalazine),thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam,piroxicam, tenoxicam), ε-acetamidocaproic acid, s-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene,nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone,superoxide dismutase, tenidap, and zileuton.

Examples of antiviral agents include interferon gamma, zidovudine,amantadine hydrochloride, ribavirin, acyclovir, valciclovir,dideoxycytidine, phosphonoformic acid, ganciclovir, and derivativesthereof

Examples of antioxidant agents include ascorbate, alpha-tocopherol,mannitol, reduced glutathione, various carotenoids, cysteine, uric acid,taurine, tyrosine, superoxide dismutase, lutein, zeaxanthin,cryotpxanthin, astazanthin, lycopene, N-acetyl-cysteine, carnosine,gamma-glutamylcysteine, quercitin, lactoferrin, dihydrolipoic acid,citrate, Ginkgo Biloba extract, tea catechins, bilberry extract,vitamins E or esters of vitamin E, retinyl palmitate, and derivativesthereof. Other therapeutic agents include squalamine, carbonic anhydraseinhibitors, alpha-2 adrenergic receptor agonists, antiparasitics,antifungals, and derivatives thereof.

The exact dose of each component administered will, of course, differdepending on the specific components prescribed, on the subject beingtreated, on the severity of the disease, for example, severity of theinflammatory reaction, on the manner of administration and on thejudgment of the prescribing physician. Thus, because ofpatient-to-patient variability, the dosages given above are a guidelineand the physician may adjust doses of the compounds to achieve thetreatment that the physician considers appropriate.

As will be understood by the skilled artisan, for HRS-Fc conjugateformulations where the carrier includes a gel-forming polymer, incertain formulations the inclusion of salt(s), in particular salinesolution, is contraindicated as inclusion of salt may either cause thesolution to gel prior to topical administration, as with certain in situgel-forming polymers (e.g., gellan gel), or the inclusion of salts mayinhibit the gelling properties of the gel-forming polymer. The skilledartisan will be able to select appropriate combinations based on thedesired properties of the formulation and characteristics of gel-formingpolymers known in the art.

Suitable aqueous saline solutions will be understood by those of skillin the art and may include, for example, solutions at a pH of from aboutpH 4.5 to about pH 8.0. In further variations of aqueous solutions(where water is included in the carrier), the pH of the formulation isbetween any of about 6 and about 8.0; between about 6 and about 7.5;between about 6 and about 7.0; between about 6.2 and about 8; betweenabout 6.2 and about 7.5; between about 7 and about 8; between about 6.2and about 7.2; between about 5.0 and about 8.0; between about 5 andabout 7.5; between about 5.5 and about 8.0; between about 6.1 and about7.7; between about 6.2 and about 7.6; between about 7.3 and about 7.4;about 6.0; about 7.1; about 6.2; about 7.3; about 6.4; about 6.5; about6.6; about 6.7; about 6.8; about 6.9; about 7.0; about 7.1; about 7.2;about 7.3; about 7.4; about 7.5; about 7.6; or about 8.0. In somevariations, the HRS-Fc conjugate formulation has a pH of about 6.0 toabout 7.0. In some variations, the formulation has a pH of about 7.4. Inparticular variations, the formulation has a pH of about 6.2 to about7.5.

In certain embodiments the concentration of the salt (e.g., NaCl) willbe, for example, from about 0% to about 0.9% (w/v). For example, theconcentration of salt may be from about 0.01 to about 0.9%, from about0.02% to about 0.9%, from about 0.03% to about 9%, from about 0.05% toabout 0.9% from about 0.07% to about 0.9%, from about 0.09% to about0.9%, from about 0.1% to about 0.9% from about 0.2% to about 0.9%, fromabout 0.3% to about 0.9%, from about 0.4% to about 0.9% from about 0.5%to about 0.9%, from about 0.6% to about 0.9%, from about 0.7% to about0.9%, from about 0.8% to about 0.9%, about 0.9%, about 0%, about 0.05%,about 0.01%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, or about 0.8%. In certainembodiments, the aqueous saline solution will be isotonic (e.g., NaClconcentration of about 0.9% NaCl (w/v)). In certain embodiments, theaqueous solution will contain a NaCl concentration of about 0.5%, about0.7%, about 0.8%, about 0.85, or about 0.75%. As will be appreciated theskilled artisan, depending on the concentrations of other components,for example where the HRS-Fc conjugates are present as salts of, theconcentration of NaCl or other salt needed to achieve an formulationsuitable for administration may vary.

In some embodiments, where the formulation is substantially free ofviscosity-increasing agents, the formulation may be substantially freeof viscosity-increasing agents such as, but not limited to polyanionicpolymers, water soluble cellulose derivatives (e.g., hypromellose (alsoknown as HPMC, hydroxypropylmethyl cellulose, andhydroxypropylcellulose), hydroxyethylcellulose, carboxmethylcellulose,etc.), polyvinyl alcohol, polyvinyl pyrrolidone, chondroitin sulfate,hyaluronic acid, soluble starches, etc. In some variations, theformulation does not incorporate a hydrogel or other retention agent(e.g., such as those disclosed in U.S. Pat. Pub. No. 2005/0255144(incorporated by reference herein in its entirety)), e.g., where thehydrogel may include hydrogels incorporating homopolymers; copolymers(e.g., tetrapolymers of hydroxymethylmethacrylate, ethylene glycol,dimethylmethacrylate, and methacrylic acid), copolymers of trimethylenecarbonate and polyglycolicacid, polyglactin 910, glyconate,poly-p-dioxanone, polyglycolic acid, polyglycolic acid felt,poly-4-hydroxybutyrate, a combination of poly(L-lactide) andpoly(L-lactide-co-glycolide), glycol methacrylate, poly-DL-lactide, orPrimacryl); composites of oxidized regenerated cellulose, polypropylene,and polydioxanone or a composite of polypropylene and poligelcaprone;etc. In some variations, the formulations do not include one or more ofpolyvinyl alcohol, hydroxypropyl methylcellulose, polyethylene glycol400 castor oil emulsion, carboxymethylcellulose sodium, propyleneglycol, hydroxypropyl guar, carboxymethylcelluose sodium, whitepetrolatum, mineral oil, dextran 70, glycerin, hypromellose, flaxseedoil, fish oils, omega 3 and omega 6 fatty acids, lutein, or primroseoil. In some variations, the formulations do not include one or more ofthe carriers described in U.S. Pat. No. 4,888,354 (incorporated byreference herein in its entirety), e.g., such as one or more of oleicacid, ethanol, isopropanol, glycerol monooleate, glycerol diooleate,methyl laurate, propylene glycol, propanol or dimethyl sulfoxide. Insome variations, the formulations are substantially free of glyceroldiooleate and isopropanol.

In particular embodiments, the gel-forming polymer may be, for example,a polysaccharide. In certain embodiments, the polysaccharide is gellangum. Gellan gum refers to a heteropolysaccharide elaborated by thebacterium Pseudomonas elodea, though the name “gellan gum” is morecommonly used in the field. Gellan gum, in particular the formulationGELRITE® is described in detail in U.S. Pat. No. 4,861,760 (herebyincorporated by reference in its entirety), in particular in its use informulation of timolol. GELRITE®, a low acetyl clarified grade of gellangum, is commercially available from Merck & Co (Rahway, N.J.) and gellangum can be commercially obtained from, among others CPKelco (Atlanta,Ga.). The preparation of polysaccharides such as gellan gum is describedin, for example, U.S. Pat. Nos. 4,326,053 and 4,326,052, which arehereby incorporated by reference in their entirety.

In certain embodiments, the gel-forming polymer is present at aconcentration of from about 0.03% to about 2% (w/v). In someembodiments, the gel-forming polymer is present at a concentration fromabout 0.03% to about 1.75%; from about 0.03% to about 1.5%, from about0.03% to about 1.25%, from about 0.03% to about 1%, from about 0.03% toabout 0.9%, from about 0.03% to about 0.8%, from about 0.03% to about0.7%, from about 0.03% to about 0.6%, from about 0.03% to about 0.5%,from about 0.05% to about 2%, from about 0.05% to about 1.75%; fromabout 0.05% to about 1.5%, from about 0.05% to about 1.25%, from about0.05% to about 1%, from about 0.05% to about 0.9%, from about 0.05% toabout 0.8%, from about 0.05% to about 0.7%, from about 0.05% to about0.6%, from about 0.05% to about 0.5%, from about 0.1% to about 2%, fromabout 0.1% to about 1.75%; from about 0.1% to about 1.5%, from about0.1% to about 1.25%, from about 0.1% to about 1%, from about 0.1% toabout 0.9%, from about 0.1% to about 0.8%, from about 0.1% to about0.7%, from about 0.1% to about 0.6%, from about 0.1% to about 0.5%, fromabout 0.2% to about 2%, from about 0.2% to about 1.75%; from about 0.2%to about 1.5%, from about 0.2% to about 1.25%, from about 0.2% to about1%, from about 0.2% to about 0.9%, from about 0.2% to about 0.8%, fromabout 0.2% to about 0.7%, from about 0.2% to, about 0.6%, from about0.2% to about 0.5%, or from about 0.5% to about 1.5%. In someembodiments, the concentration of gel-forming polymer is about 0.1%,about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%.

In particular embodiments, the gel-forming polymer is gellan gum at aconcentration of from about 0.05% to about 2% (w/v), from about 0.1% toabout 2% (w/v), from about 0.1% to about 1% (w/v), from about 0.05% toabout 1% (w/v) or from about 0.1% to about 0.6% (w/v). In someembodiments, the concentration of gellan gum is about 0.1%, about 0.2%,about 0.4%, about 0.6%, about 0.8%, about 1%.

In some embodiments of the formulations, the formulation may includeadditional components such as one or more preservatives, one or moresurfactants, or one or more pharmaceutical agents. In particularembodiments, the formulation may include additional components such asone or more preservatives, one or more surfactants, one or more tonicityagents, one or more buffering agents, one or more chelating agents, oneor more viscosity-increasing agents, one or more salts, or one or morepharmaceutical agents. In certain of these embodiments, the formulationmay include (in addition to a HRS-Fc conjugate (or a pharmaceuticallyacceptable salt thereof) and carrier): one or more preservatives, one ormore buffering agents (e.g., one, two, three, etc.), one or morechelating agents, and one or more salts. In some embodiments, theformulation may include (in addition to a HRS-Fc conjugate (or apharmaceutically acceptable salt thereof) and carrier): one or morepreservatives, one or more tonicity agents, one or more bufferingagents, one or more chelating agents, and one or moreviscosity-increasing agents.

In some embodiments, the viscosity of the formulation is about the sameas the viscosity of a saline solution containing the same concentrationof a HRS-Fc conjugate (or a pharmaceutically acceptable salt thereof).In some embodiments, the formulation is substantially free ofgel-forming polymers. In certain embodiments, where the carrier iswater, the formulation may additionally include one or more chelatingagents (e.g., EDTA disodium (EDTA), one or more preservatives (e.g.,benzalkonium chloride, benzethonium chloride, chlorhexidine,chlorobutanol, methylparaben, phenylethyl alcohol, propylparaben,thimerosal, phenylmercuric nitrate, phenylmercuric borate,phenylmercuric acetate, or combinations of two or more of theforegoing), salt (e.g., NaCl) and one or more buffering agents (e.g.,one or more phosphate buffers (e.g., dibasic sodium phosphate, monobasicsodium phosphate, combinations thereof, etc.), citrate buffers, maleatebuffers, borate buffers, and combination of two or more of theforegoing.).

In particular embodiments, the chelating agent is EDTA disodium, thepreservative is benzalkonium chloride, the salt is NaCl, and thebuffering agents are dibasic sodium phosphate and monobasic sodiumphosphate. In certain of these embodiments, the formulation issubstantially free of polymer. In some embodiments, the formulation issubstantially free of substantially viscosity-increasing agent(s) (e.g.,carboxymethylcellulose, polyanionic polymers, etc.). In someembodiments, the viscosity of the formulation is about the same as theviscosity of a saline solution containing the same concentration of aHRS-Fc conjugate (or a pharmaceutically acceptable salt thereof). Insome of these embodiments, the concentration of a HRS-Fc conjugate (or apharmaceutically acceptable salt thereof) if from about 0.02% to about3%, from about 0.02% to about 2%, from about 0.02% to about 1% (w/v). Incertain embodiments, the concentration of a HRS-Fc conjugate (or apharmaceutically acceptable salt thereof), is about 0.01%, about 0.02%,about 0.03%, about 0.05%, about 0.07%, about 0.1%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.8% or about 1% (w/v).

In certain embodiments, where the carrier includes water, aviscosity-increasing agent may also be included in the formulation. Theskilled artisan will be familiar with viscosity-increasing agents thatare suitable (e.g., water-soluble cellulose derivatives (e.g.,hypromellose (also known as HPMC, hydroxypropylmethyl cellulose, andhydroxypropylcellulose), hydroxyethylcellulose, carboxmethylcellulose,etc.), polyvinyl alcohol, polyvinyl pyrrolidone, chondroitin sulfate,hyaluronic acid, and soluble starches. It is intended that whenviscosity-increasing agents are used, they are not included in highenough concentrations such that the formulation would form a gel priorto or after administration (e.g., wherein the concentration of theviscosity-increasing agent is not sufficient to induce gel formation).

While exact concentrations of viscosity-increasing agents will dependupon the selection and concentration of other components in theformulation as well as the particular viscosity-increasing agent(s)selected, in general, viscosity-increasing agents may be present in aconcentration such that the viscosity of the resulting solution is lessthan about 1000 centipoise. In certain embodiments, the viscosity of theformulation is less than about 900, less than about 800, less than about700, less than about 600, less than about 500, less than about 400, lessthan about 300, less than about 200, less than about 150, less thanabout 100, less than about 50 centipoise. In some embodiments, theviscosity of the formulation is about 200, about 150, about 100, about50 centipoise. In particular embodiments, the viscosity is less thanabout 200 centipoise. In others, less than about 120 centipoise or lessthan about 100 centipoise. In some embodiments, the viscosity is about100 centipoise. In others about 50 centipoise. In still otherembodiments the viscosity is about 200 centipoise. Methods for measuringviscosity are well known to the skilled artisan. For example, asdescribed in United States Pharmacopoeia 29 (Chapter 911) Viscosity,page 2785 (which is herein incorporated by reference in its entirety).As is well known to the skilled artisan, formulations commonlyconsidered “gels” will have viscosity significantly greater than 1000centipoise, for example, greater than about 2000 centipoise, greaterthan about 5000 centipoise.

In some embodiments, including (but not limited to) where the use ofsalts is contraindicated as described above, the formulation may furtherinclude one or more tonicity agents. As used herein, the term “tonicityagent” and its cognates refers to agents that adjust the tonicity of theformulation, but are not salts (e.g., not NaCl), which, as will beappreciated by the skill artisan in view of the teaching providedherein, are contraindicated for some formulations due to the presence ofcertain of the gel-forming polymers or viscosity-increasing agents.These agents may be used to prepare formulations that are isotonic ornear isotonic (e.g., somewhat hyper- or hypo-isotonic; e.g., withinabout ±20%, about ±15%, about ±10%, about ±5% of being isotonic).Tonicity agent(s) may also be used in formulations where the use ofsalts is not contraindicated.

Tonicity agents that may be used to adjust the tonicity of formulationthe formulations described herein and are known to the skilled artisanand can be selected based on the teaching provided herein. For example,tonicity agents include polyols (e.g., sugar alcohols (e.g., mannitol,etc.), trihydroxy alcohols (e.g., glycerin, etc.), propylene glycol orpolyethylene glycol, etc.), or combinations of two or more polyols.Likewise, the concentration of the tonicity agent(s) will depend uponthe identity and concentrations of the other components in theformulation and can be readily determined by the skilled artisan in viewof the teaching provided herein.

In certain embodiments, the tonicity agent is glycerin or mannitol. Insome embodiments, the tonicity agent is glycerin. In other embodimentsit is, mannitol. In still others a combination of mannitol and glycerinmay be used. Exemplary concentrations of tonicity agents include, forexample from about 0.001 to about 3%. In some embodiments, theconcentration of the tonicity agent (e.g., mannitol or glycerin) is, forexample, about 0.001% to about 2.7%, about 0.001% to about 2.5%, about0.001% to about 2%, about 0.001% to about 1.5%, about 0.001% to about1%, about 0.01% to about 3%, about 0.01% to about 2.7%, about 0.01% toabout 2.5%, about 0.01% to about 2%, about 0.01% to about 1.5%, about0.01% to about 1%, about 0.1% to about 3%, about 0.1% to about 2.7%,about 0.1% to about 2.5%, about 0.1% to about 2%, about 0.1% to about1.5%, about 0.1% to about 1%, about 0.01% about 1% to about 3%; about 1%to about 2.5%; about 1% to about 2%; about 1% to about 1.8%; about 1% toabout 1.5%; or about 0.001%, about 0.01%, about 0.05%, about 0.08%,about 0.1%, about 0.2%, about 0.5%, about 0.8%, about 1%, about 1.5%,about 1.8%, about 2%, about 2.2%, about 2.5%, about 2.8%, or about 3%(w/v). In certain embodiments, the tonicity agent is mannitol. In someof these embodiments, the carrier includes a gel-forming agent (e.g.,gellan gum).

In some embodiments, the tonicity agent is mannitol. In certain of theseembodiments, the carrier includes a viscosity-increasing agent (e.g.,water soluble cellulose derivatives (e.g., hypromellose), polyvinylalcohol, polyvinyl pyrrolidone, chondroitin sulfate, hyaluronic acid, orsoluble starches).

In some embodiments, the formulation may additionally include apreservative (e.g., benzalkonium chloride, benzethonium chloride,chlorhexidine, chlorobutanol, methylparaben, Phenylethyl alcohol,propylparaben, thimerosal, phenylmercuric nitrate, phenylmercuricborate, or phenylmercuric acetate, peroxides), or a combination of twoor more of the foregoing preservatives. In certain embodiments, thepreservative is benzalkonium chloride.

As will be appreciated by the skilled artisan, preservatives may bepresent in concentrations of from about 0.001% to about 0.7% (w/v). Inparticular embodiments, the preservative(s) may be present in aconcentration of from about 0.001% to about 0.5% (w/v); from about0.001% to about 0.05% (w/v), from about 0.001% to about 0.02% (w/v),from about 0.001% to about 0.015% (w/v), from about 0.001% to about0.005% (w/v), from about 0.01% to about 0.02%, from about 0.002% toabout 0.01%, from about 0.015% to about 0.05%, less than about <0.5%,from about 0.005% to about 0.01%, from about 0.001% to about 0.15%, fromabout 0.002% to about 0.004%, from about 0.001% to about 0.002%. In someembodiments the concentration of the preservative may be, for example,about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.03%, about0.05%, about 0.1%, about 0.2%, about 0.5%, or about 0.7% (w/v). Typicalconcentrations (w/v) for various commonly used preservatives are listedin Table C below.

TABLE C Approximate Concentration Preservative Range (w/v) Benzalkoniumchloride  0.01-0.02% Benzethonium chloride  0.01-0.02% Chlorhexidine0.002-0.01% Chlorobutanol     <0.5% Methylparaben 0.015-0.05%Phenylethyl alcohol     <0.5% Propylparaben 0.005-0.01% Thimerosal0.001-0.15% Phenylmercuric nitrate 0.002-0.004%  Phenylmercuric borate0.002-0.004 Phenylmercuric acetate 0.001-0.002

In certain embodiments, the formulation may additionally include asurfactant, or combinations of two or more surfactants. In particularembodiments, the formulation is substantially free of surfactant. Asused herein, the term “substantially free” is intended to refer tolevels of a particular component that are undetectable using routinedetection methods and protocols known to the skilled artisan. Forexample, HPLC (including chiral HPLC, chiral HPLC/MS, LC/MS/MS etc.),thin layer chromatography, mass spectrometry, polarimetry measurements,gas-chromatography-mass spectrometry, or others.

In particular embodiments, the formulation may further include achelating agent (e.g., EDTA disodium (EDTA) (e.g., EDTA disodium(dihydrate), etc.) citrates, etc.). In some embodiments, a combinationof chelating agents may be present. As will be appreciated by those ofskill in the field, chelating agents can be used to hinder degradationof the formulation components and thereby increase the shelf life offormulations. As will be appreciated by the skilled artisan, use of EDTAin combination with gellan gum formulation may be contraindicated as theEDTA can cause gel formation prior to administration of the gellan gumformulation.

Typical concentrations for chelating agents are from about 0.005% to0.1% (w/v). For example, from about 0.005% to about 0.09%, from about0.005% to about 0.08%, from about 0.005% to about 07%, from about0.005%, to about 0.06%, from about 0.005% to about 0.05%, from about0.005 to about 0.04%, from about 0.005% to about 0.03%, from about 0.01%to about 0.1%, from about 0.01% to about 0.09%, from about 0.01% toabout 0.08%, from about 0.01% to about 0.07%, from about 0.01% to about0.06%, from about 0.01% to about 0.05%, from about 0.01% to about 0.04%,etc. In certain embodiments, the concentration of chelating agent(s) isabout 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.05%, about0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%.

In particular embodiments, the chelating agent is EDTA disodium. Incertain embodiments, the chelating agent is EDTA disodium (dihydrate).In some of these embodiments, the EDTA disodium dihydrate is present ata concentration of about 0.01% (w/v).

In some embodiments, the formulation may additionally include one ormore buffering agents (e.g., phosphate buffer(s) (e.g., sodium phosphatebuffers (e.g., dibasic sodium phosphate, monobasic sodium phosphate,etc.), citrate buffers, maleate buffers, borate buffers, etc.). As willbe appreciated by the skilled artisan, the one or more bufferingagent(s) should be selected in combination with the other components ofa given formulation to achieve a pH suitable for use (e.g., pH of about4.5 to about 8).

In certain embodiments, the buffering agent is a phosphate buffer orcombination of two or more phosphate buffers. In certain embodiments,the buffering agents are dibasic sodium phosphate and monobasic sodiumphosphate.

Typical concentrations for buffering agent(s) for example, phosphatebuffering agent(s) may be from about 0.005 molar to 0.1 molar. In someembodiments, the buffering agent(s) may be at a concentration of about0.01 to about 0.1, from about 0.01 to about 0.08, from about 0.01 toabout 0.05, from about 0.01 to about 0.04, from about 0.02 to about 0.1,from about 0.02 to about 0.08, from about 0.02 to about 0.06, from about0.02 to about 0.05, from about 0.02 to about 0.04 molar, etc. Inparticular embodiments, there are two buffering agents. Exemplarybuffering agents include a combination of dibasic sodium phosphate(e.g., dibasic sodium phosphate.7H₂O) and monobasic sodium phosphate(e.g., monobasic sodium phosphate anhydrous). In some embodiments, theconcentration of the buffering agent(s) is about 0.005 molar, about 0.01molar, about 0.02 molar, about 0.03 molar, about 0.04 molar, about 0.05molar, about 0.06 molar, about 0.07 molar, or about 0.1 molar.

An additional aspect of the invention includes use of the formulationsas described herein in the manufacture of a medicament. Particularly,the manufacture of a medicament for use in the treatment and/orprevention of conditions as described herein. Further, the formulations,variously described herein, are also intended for use in the manufactureof a medicament for use in treatment and/or prevention of the conditionsand, in accordance with the methods, described herein, unless otherwisenoted.

Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., 19th Edition (1995). The compositionsand agents provided herein may be administered according to the methodsof the present invention in any therapeutically effective dosingregimen. The dosage amount and frequency are selected to create aneffective level of the agent without harmful effects. The effectiveamount of a compound of the present invention will depend on the routeof administration, the type of warm-blooded animal being treated, andthe physical characteristics of the specific warm-blooded animal underconsideration. These factors and their relationship to determining thisamount are well known to skilled practitioners in the medical arts. Thisamount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays have beendescribed e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (eachspecifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

In certain embodiments, the agents provided herein may be attached to apharmaceutically acceptable solid substrate, including biocompatible andbiodegradable substrates such as polymers and matrices. Examples of suchsolid substrates include, without limitation, polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such aspoly(lactic-co-glycolic acid) (PLGA) and the LUPRON DEPOT™ (injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, collagen, metal,hydroxyapatite, bioglass, aluminate, bioceramic materials, and purifiedproteins.

In one particular embodiment, the solid substrate comprises Atrigel™(QLT, Inc., Vancouver, B.C.). The Atrigel® drug delivery system consistsof biodegradable polymers dissolved in biocompatible carriers.Pharmaceuticals may be blended into this liquid delivery system at thetime of manufacturing or, depending upon the product, may be added laterby the physician at the time of use. When the liquid product is injectedinto the subcutaneous space through a small gauge needle or placed intoaccessible tissue sites through a cannula, water in the tissue fluidscauses the polymer to precipitate and trap the drug in a solid implant.The drug encapsulated within the implant is then released in acontrolled manner as the polymer matrix biodegrades with time.

In particular embodiments, the amount of a HRS-Fc conjugate compositionadministered will generally range from a dosage of from about 0.1 toabout 100 mg/kg, and typically from about 0.1 to 10 or 20 mg/kg whereadministered orally, subcutaneously, or intravenously. In particularembodiments, a dosage is about 1 mg/kg, about 3 mg/kg, about 5 mg/kg,about 7.5 mg/kg, or about 10 mg/kg. In certain embodiments, acomposition is administered in a single dosage of 0.1 to 10 mg/kg or 0.5to 5 mg/kg. In some embodiments, a composition is administered in adosage of 0.1 to 50 mg/kg, 0.5 to 20 mg/kg, or 5 to 20 mg/kg.

For humans, the daily dosage used may range from, about 0.1 mg/kg to 0.5mg/kg, about 1 mg/kg to 5 mg/kg, about 5 mg/kg to 10 mg/kg, about 10mg/kg to 20 mg/kg, about 20 mg/kg to 30 mg/kg, about 30 mg/kg to 50mg/kg, and about 50 mg/kg to 100 mg/kg/24 hours.

For HRS-Fc conjugates with longer half lives, the human dosage used mayrange, for example, from about 0.1 mg/kg/week to 0.5 mg/kg/week, about 1mg/kg/week to 5 mg/kg/week, about 5 mg/kg/week to 10 mg/kg/week, about10 mg/kg/week to 20 mg/kg/week, about 20 mg/kg/week to 30 mg/kg/week,about 30 mg/kg/week to 50 mg/kg/week, or about 50 mg/kg/week to 100mg/kg/week.

HRS-Fc conjugates with still longer half lives may be dosed in humansabout 0.1 mg/kg/month to 0.5 mg/kg/month, about 1 mg/kg/month to 5mg/kg/month, about 5 mg/kg/month to 10 mg/kg/month, about 10 mg/kg/monthto 20 mg/kg/month, about 20 mg/kg/month to 30 mg/kg/month, about 30mg/kg/month to 50 mg/kg/month, or about 50 mg/kg/month to 100mg/kg/month.

In various embodiments, the dosage is about 50-2500 mg per day, 100-2500mg/day, 300-1800 mg/day, or 500-1800 mg/day, or 500-2500 mg per week,1000-2500 mg/week, 300-1800 mg/week, or 500-1800 mg/week, or 500-2500 mgper month, 1000-2500 mg/month, 300-1800 mg/month, or 500-1800 mg/month.In some embodiments, the dosage is between about 100 to 600 mg/day, 100to 600 mg/week, or 100 to 600 mg/month. In some embodiments, the dosageis between about 300 and 1200 mg/day, 300 and 1200 mg/week, or 300 and1200 mg/month. In particular embodiments, the composition or agent isadministered at a dosage of 100 mg/week, 2.4 mg/week 300 mg/week, 600mg/week, 1000 mg/week, 1200 mg/week or 1800 mg/week, in one or moredoses per week or per month (i.e., where the combined doses achieve thedesired weekly or monthly dosage). In some embodiments, a dosage is 100mg bid, 150 mg bid, 240 mg bid, 300 mg bid, 500 mg bid, or 600 mg bid.In various embodiments, the composition or agent is administered insingle or repeat dosing. The initial dosage and subsequent dosages maybe the same or different.

In some embodiments, total daily dose may be about 0.001 mg, about 0.005mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, 0.5 mg, 1 mg, about 2mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg,about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg or about100 mg/24 hours.

In some embodiments, total weekly dose may be about 0.001 mg, about0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, 0.5 mg, 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg orabout 100 mg/week.

In some embodiments, total monthly dose may be about 0.001 mg, about0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, 0.5 mg, 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg orabout 100 mg/month.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of these and other therapies(e.g., ex vivo therapies) can be readily monitored by conventionalmethods and assays and based on criteria known to the physician or otherpersons of skill in the art.

It will be further appreciated that for sustained delivery devices andcompositions the total dose of HRS contained in such delivery systemwill be correspondingly larger depending upon the release profile of thesustained release system. Thus, a sustained release composition ordevice that is intended to deliver HRS-Fc conjugates over a period of 5days will typically comprise at least about 5 to 10 times the daily doseof HRS-Fc conjugate; a sustained release composition or device that isintended to deliver a HRS-Fc conjugate over a period of 365 days willtypically comprise at least about 400 to 800 times the daily dose of theHRS-Fc conjugate (depending upon the stability and bioavailability ofthe HRS-Fc conjugate when administered using the sustained releasesystem).

In certain embodiments, a composition or agent is administeredintravenously, e.g., by infusion over a period of time of about, e.g.,10 minutes to 90 minutes. In other related embodiments, a composition oragent is administered by continuous infusion, e.g., at a dosage ofbetween about 0.1 to about 10 mg/kg/hr over a time period. While thetime period can vary, in certain embodiments the time period may bebetween about 10 minutes to about 24 hours or between about 10 minutesto about three days.

In particular embodiments, an effective amount or therapeuticallyeffective amount is an amount sufficient to maintain a concentration ofthe HRS-Fc conjugate in the blood plasma of a subject above about 300pM, above about 1 nM, above about 10 nM, above about 100 nM, or aboveabout 1000 nM.

In certain embodiments, an IV or SC dosage is an amount sufficient toachieve a blood plasma concentration (C_(max)) of between about 1,000 nMto about 5,000 nM or between about 200 nM to about 1,000 nM, or about 20nM to about 200 nM.

In particular embodiments, a HRS-Fc conjugate is administered in anamount and frequency sufficient to achieve in the mammal a blood plasmaconcentration having a mean trough concentration of between about 300 pMand about 1 nM and/or a steady state concentration of between about 300pM and about 1 nM. In some embodiments, the C_(min) of the HRS-Fcconjugate in the blood plasma of the mammal is maintained above about 1nM and/or a steady state concentration of between about 1 nM and about10 nM. In certain embodiments, the C_(min) of the HRS-Fc conjugate inthe blood plasma of the mammal is maintained above about 10 nM and/or asteady state concentration of between about 10 nM and about 100 nM. Incertain embodiments, the C_(min) of the HRS-Fc conjugate in the bloodplasma of the mammal is maintained above about 100 nM and/or a steadystate concentration of between about 100 nM and about 1000 nM.

In particular embodiments of the present invention, an effective amountof the HRS-Fc conjugate, or the blood plasma concentration of the HRS-Fcconjugate, is achieved or maintained, with a single administration,e.g., for at least 15 minutes, at least 30 minutes, at least 45 minutes,at least 60 minutes, at least 90 minutes, at least 2 hours, at least 3hours, at least 4 hours, at least 8 hours, at least 12 hours, at least24 hours, at least 48 hours, at least 3 days, at least 4 days, at least5 days, at least 6 days, at least one week, at least 2 weeks, at leastone month, at least 2 months, at least 3 months, at least 4 months, orat least 6 months.

In particular embodiments, the effective dosage achieves the bloodplasma levels or mean trough concentration of a composition or agentdescribed herein. These may be readily determined using routineprocedures.

Embodiments of the present invention, in other aspects, provide kitscomprising one or more containers filled with one or more of the HRS-Fcconjugates, polypeptides, polynucleotides, antibodies, multiunitcomplexes, compositions thereof, etc., of the invention, as describedherein. The kits can include written instructions on how to use suchcompositions (e.g., to modulate cellular signaling, angiogenesis,cancer, inflammatory conditions, diagnosis etc.).

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated, or for the desired diagnostic application. An additionaltherapeutic agent may be contained in a second container, if desired.Examples of additional therapeutic agents include, but are not limitedto anti-neoplastic agents, anti-inflammatory agents, antibacterialagents, antiviral agents, angiogenic agents, etc.

The kits herein can also include one or more syringes or othercomponents necessary or desired to facilitate an intended mode ofdelivery (e.g., stents, implantable depots, etc.).

Certain embodiments of the present invention now will be illustrated bythe following Examples. The present invention may, however, be embodiedin many different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

EXAMPLES Example 1 Production of His Tagged Resokine (HRS ComprisingAmino Acids 1-60)

Codon optimization and gene synthesis. DNA encoding Resokine (HRS(1-60))was codon-optimized for E. coli expression using the algorithm developedby DNA2.0 (Menlo Park, Calif.).

The codon-optimized DNA sequence is as follows:

(SEQ ID NO: 261) ATGGCAGAACGTGCGGCATTGGAAGAATTGGTTAAACTGCAAGGTGAACGTGTTCGTGGTCTGAAGCAGCAGAAGGCTAGCGCGGAGCTGATCGAAGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAACACCACCATCACCATCAC

The translated protein sequence is as follows:

(SEQ ID NO: 262) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKHHHHHH 

Additionally, engineered versions of this construct were prepared withcysteine residues inserted close to the N-terminus (comprisingadditional N-terminal Met and Cys residues), C-terminus (comprising anadditional C-terminal cysteine at position 61), and in the linker domainjoining the 2 alpha helical sections of the molecule (comprising themutation Ala 26→Cys). The codon optimized DNA sequences, andcorresponding amino acid sequences for these constructs are listedbelow.

H-N4: 1-H (codon-HRS(1-60)-M1MC-6×His):

(SEQ ID NO: 263) ATGTGTGCAGAAAGAGCCGCCCTGGAAGAGTTAGTTAAGTTGCAAGGTGAACGTGTCCGTGGTCTGAAGCAGCAGAAGGCTAGCGCGGAGCTGATCGAAGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAACACCACCATCACCATCA C

The translated protein sequence is as follows:

(SEQ ID NO: 264) MCAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKHHHHHH H-N4:2-H (codon-HRS(1-60)-A26C-6xHis):(SEQ ID NO: 265) ATGGCAGAACGTGCGGCATTGGAAGAATTGGTTAAACTGCAAGGTGAACGTGTTCGTGGTCTGAAGCAGCAGAAGTGCAGCGCGGAGCTGATCGAAGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAACACCACCATCACCATC AC

The translated protein sequence is as follows:

(SEQ ID NO: 266) MAERAALEELVKLQGERVRGLKQQKCSAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKHHHHHH H-N4:3-H (codon-HRS(1-60)-C61-6xHis): (SEQ ID NO: 267)ATGGCAGAACGTGCGGCATTGGAAGAATTGGTTAAACTGCAAGGTGAACGTGTTCGTGGTCTGAAGCAGCAGAAGGCTAGCGCGGAGCTGATCGAAGAAGAGGTGGCCAAACTGCTGAAGCTGAAGGCGCAGCTGGGCCCGGACGAGAGCAAACAAAAGTTCGTCCTGAAAACCCCGAAATGCCACCACCATCACCATCA C

The translated protein sequence is as follows:

(SEQ ID NO: 268) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKCHHHHHH

The corresponding genes were synthesized (DNA 2.0) with a C-terminal6×His tag and subcloned into the pJexpress411 expression vector wherethe T7 promoter was used to drive the transcription and kanamycinresistance was used for antibiotic selection.

Expression Strain.

BL21(DE3) competent cells (Novagen, cat.

no. 69450) were transformed with the relevant codon-optimized expressionconstruct, as described. Briefly, the plasmid (1 μL) was added into 50μL of the competent cells. The reaction was mixed and incubated on icefor 30 minutes. The reaction was heat-shocked for at 42° C. for 30seconds followed by a cold-shock on ice for 2 minutes. Then the SOCmedium (500 μL) was added and the tube was incubated at 37° C., 250 rpmfor 1 hour. Finally, an aliquot of the culture (50 μL) was spread on theKanamycin plate (Teknova S9641) and incubated at 37° C. overnight. Asingle colony was picked and used for expression scale-up.

Medium.

M9YE medium was prepared by mixing 200 mL sterile M9 minimal salt 5×(BD248510), 778 mL of 30 g/L yeast extract in sterile purified water(BD212750), 20 mL sterilized 20% glucose (Sigma G7021) and 2 mL sterile1.0 M MgSO4 (Sigma M7506). The feeding solution contains 5% yeastextract, 50% glucose, trace elements and 2 g/L magnesium sulfate.Kanamycin sulfate (Invitrogen 15160) was added to a final concentrationof 100 μg/mL in both M9YE and feeding solution.

Fed-Batch Fermentation.

A 4 L fermentor (Sartorius Biostat B plus) with MFCS/DA software wasused for the fed-batch fermentation. The agitation was set at 1000 rpm.The pH value was controlled at 7.0 automatically by the addition of 30%ammonium hydroxide (Sigma 221228) and 30% phosphoric acid (Sigma P5811).The air was provided at a flow rate of 4 L/min with an oil-freediaphragm air compressor (Cole-Parmer). The air was passed through a 0.2μm Midisart 2000 filter (Sartorius 17805). The pure oxygen (West Air)was supplied automatically to control the dissolved oxygen level at 70%.The temperature was controlled at 30° C. with a Neslab RTE7 circulator(Thermo Scientific). The foaming was controlled by addition of theantifoam 204 (Sigma A8311). The initial volume of M9YE medium in thefermentor was 3L. The fermentor was inoculated with 150 mL of the seedculture grown overnight at 30° C. and 250 rpm. When the glucose wasdepleted in the vessel, the concentrated feeding solution was introducedinto the vessel by a peristaltic pump set at 0.9 mL/min. When theoptical density of the cells at 600 nm reached about 30, the culture wasinduced with 0.5 mM IPTG (Fisher Scientific BP1755). The culture was runovernight (about 18-hour fed-batch phase) and harvested bycentrifugation at 6,000×g for 1 hour. The cell pellet was stored at −20°C. until purification. The expression of Resokine was confirmed on theSDS-PAGE.

Purification of Proteins.

Resokine and the Cys variants thereof were purified from E. coli cellpaste through cell lysis and clarification; immobilized metal affinitychromatography, and cation exchange chromatography. Frozen cellsweighing 10 g and containing Resokine or Cys variants were thawed andresuspended in 1× NiNTA buffer (50 mM Tris, 0.3 M NaCl 25 mM imidazolepH 8) at a 4:1 ml/g paste ratio along with 5 mM beta-mercaptoethanol(Sigma Cat#M7154-25ML) and 1 protease inhibitor cocktail tablet (RocheCat #05 056 489 001). After all cells were resuspended, cells were lysedon ice in a Microfluidizer M-110Y, 2 passes at 15000 psi on ice torelease soluble Resokine or Cys variants. NiNTA buffer was added afterthe second pass to flush remaining lysate through the Microfluidizer(100-120 ml final volume after lysis). Lysate was centrifuged for15000×g @ 4 C for 30 min. Supernatant was filtered 0.45/0.2 um with anAcropak 200 (Pall Cat#12094). Filtered supernatant is clarified lysate.

Immobilized Metal Affinity Chromatography (IMAC) Purification.

Clarified lysate from 10 g cell paste was loaded onto a gravity-flowcolumn containing 3 ml NiNTA resin (Qiagen #30210) and pre-equilibratedin NiNTA buffer. Resin was washed with 50 column volumes (CV) of 0.1%Triton X-114 in 1× NiNTA buffer to remove endotoxin, then 30 CV 1× NiNTAbuffer, followed by elution with 5 CV NiNTA elution buffer (50 mM Tris,0.3 M NaCl, 0.3 M imidazole pH 8 @ 4 C.

Cation Exchange (CEX) Chromatography Purification.

CEX load was prepared by diluting NiNTA eluent 1/20× in CEX A buffer (10mM sodium phosphate pH 7.0, 2 mM DTT), then loaded onto SP SepharoseHigh Performance column equilibrated in CEX A. Protein was eluted with alinear gradient of 0-100% B over 20 CV, where A=10 mM sodium phosphatepH 7.0, 2 mM DTT and B=10 mM sodium phosphate, 1 M sodium chloride 2 mMDTT pH 7.0, monitoring absorbance at 214 nm. Fractions were pooledcorresponding to the main peak in the elution gradient by absorbance at214 nm. CEX pool was buffer exchanged into 1×PBS pH 7.4 (Gibco #10010)using Amicon Ultra-15 3 kD MWCO ultracentrifugal devices.

Example 2 Production of His Tagged Full-Length Histidyl-tRNA Synthetase(Hrs)

Codon Optimization and Gene Synthesis.

The full length HisRS gene was codon-optimized for E. coli expressionand subcloned into pET21a vector where the T7 promoter was used to drivethe transcription. In addition, a 5-amino acid linker and 6×His tag wereattached to the C-terminus.

The DNA sequence is as follows:

(SEQ ID NO: 269) ATGGCGGAACGTGCCGCACTGGAAGAATTGGTTAAATTACAGGGAGAACGCGTACGTGGTCTTAAACAACAAAAAGCCTCTGCGGAATTGATTGAAGAAGAAGTTGCCAAATTACTGAAACTGAAAGCTCAACTTGGACCCGATGAAAGTAAACAAAAATTTGTGTTGAAAACGCCCAAAGGAACCCGTGATTATAGTCCACGTCAAATGGCCGTTCGTGAAAAAGTGTTCGACGTTATTATTCGCTGTTTTAAACGTCACGGTGCTGAAGTAATCGATACCCCCGTATTTGAATTGAAAGAGACTCTGATGGGCAAATATGGTGAAGATTCTAAACTGATTTATGATTTGAAAGACCAAGGAGGTGAACTGCTGAGCCTGCGCTACGACTTAACTGTGCCTTTTGCCCGTTACTTAGCCATGAATAAaTTaACCAACATCAAACGTTACCATATTGCAAAAGTATATCGCCGCGACAACCCTGCAATGACTCGTGGACGCTATCGCGAATTCTATCAGTGTGATTTTGATATTGCCGGAAATTTCGACCCGATGATCCCGGATGCCGAGTGTTTGAAAATTATGTGTGAAATTCTGAGTTCGTTGCAGATCGGAGACTTTCTTGTAAAAGTTAATGACCGCCGTATTCTGGATGGTATGTTTGCTATTTGCGGTGTTTCTGATTCCAAATTCCGTACAATCTGCTCAAGCGTGGACAAATTGGATAAAGTGTCTTGGGAAGAAGTAAAAAATGAAATGGTGGGAGAAAAAGGCCTGGCTCCAGAAGTAGCAGACCGTATTGGTGACTATGTTCAACAACATGGCGGTGTGTCCTTAGTCGAACAGTTATTACAGGATCCTAAACTGAGCCAAAATAAACAAGCACTTGAAGGACTGGGAGATCTGAAATTACTCTTTGAATATCTGACCTTATTTGGGATTGATGATAAAATTAGCTTTGATCTGAGCTTGGCCCGCGGTCTTGATTATTATACCGGCGTGATTTACGAAGCTGTTCTCTTGCAAACCCCAGCCCAGGCGGGCGAAGAGCCTTTGGGAGTCGGCAGTGTGGCAGCCGGTGGTCGTTATGATGGTTTGGTAGGAATGTTTGACCCTAAAGGCCGTAAAGTACCATGTGTGGGGCTTTCTATCGGTGTCGAACGTATCTTTTCTATTGTTGAACAACGTCTTGAAGCTTTGGAGGAAAAGATCCGTACCACGGAAacCCAAGTCTTAGTTGCaAGTGCCCAAAAAAAACTGTTAGAAGAACGCCTGAAACTCGTATCAGAACTTTGGGACGCCGGCATCAAGGCCGAACTGCTGTATAAAAAGAACCCGAAATTGTTAAACCAACTCCAGTATTGTGAAGAAGCTGGGATCCCACTCGTAGCTATTATTGGTGAGCAAGAATTAAAAGATGGCGTGATTAAACTGCGTTCAGTAACAAGCCGTGAAGAGGTAGATGTACGTCGCGAAGACTTAGTGGAAGAAATTAAACGCCGCACCGGTCAACCGTTATGTATTTGCGCGGCCGCACTCGAGCACCACCA CCACCACCACTGA

The sequence of the translated protein is as follows:

(SEQ ID NO: 270) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCICAAALEHHHHHH

Expression Strain.

The BL21(DE3) competent cells (Novagen, cat. no. 69450) were transformedwith the codon-optimized expression construct. Briefly, the plasmid (1μL) was added into 50 μL of the competent cells. The reaction was mixedand incubated on ice for 30 minutes. The reaction was heat-shocked forat 42° C. for 30 seconds followed by a cold-shock on ice for 2 minutes.Then the SOC medium (500 μL) was added and the tube was incubated at 37°C., 250 rpm for 1 hour. Finally, an aliquot of the culture (50 μL) wasspread on the Ampicillin plate (Teknova S9641) and incubated at 37° C.overnight. Single colony was picked and used for expression scale-up.

Medium.

The M9YE medium was prepared by mixing 200 mL sterile M9 minimal salt 5×(BD248510), 778 mL of 30 g/L yeast extract in sterile purified water(BD212750), 20 mL sterilized 20% glucose (Sigma G7021) and 2 mL sterile1.0 M MgSO4 (Sigma M7506). The feeding solution contains 5% yeastextract, 50% glucose, trace elements and 2 g/L magnesium sulfate.Ampicillin was added to a final concentration of 100 μg/mL in both M9YEand feeding solution.

Fed-Batch Fermentation.

A 4 L fermentor (Sartorius Biostat B plus) with MFCS/DA software wasused for the fed-batch fermentation. The agitation was set at 1000 rpm.The pH value was controlled at 7.0 automatically by the addition of 30%ammonium hydroxide (Sigma 221228) and 30% phosphoric acid (Sigma P5811).The air was provided at a flow rate of 4 L/min with an oil-freediaphragm air compressor (Cole-Parmer). The air was passed through a 0.2μm Midisart 2000 filter (Sartorius 17805). The pure oxygen (West Air)was supplied automatically to control the dissolved oxygen level at 70%.The temperature was controlled at 30° C. with a Neslab RTE7 circulator(Thermo Scientific). The foaming was controlled by addition of theantifoam 204 (Sigma A8311). The initial volume of M9YE medium in thefermentor was 3L. The fermentor was inoculated with 150 mL of the seedculture grown overnight at 30° C. and 250 rpm. When the glucose wasdepleted in the vessel, the concentrated feeding solution was introducedinto the vessel by a peristaltic pump set at 0.9 mL/min. When theoptical density of the cells at 600 nm reached about 30, the culture wasinduced with 0.5 mM IPTG (Fisher Scientific BP1755). The culture was runovernight (about 18-hour fed-batch phase) and harvested bycentrifugation at 6,000×g for 1 hour. The cell pellet was stored at −20°C. until purification. The expression of HisRS was confirmed on theSDS-PAGE.

Purification of HisRS.

Frozen cell paste (40 g) were resuspended in 160 mL (i.e. 4 mL/g cellpaste) of Lysis Buffer (20 mM Tris, 400 mM NaCl, 20 mM Imidazole, 14 mMβ-ME, pH 8.0 at 4° C.). Complete EDTA-FREE protease inhibitor tablets(Roche) were added to the suspension at a ratio of 1 tablet/50 mL. Thesuspension was passed through a microfluidizer (Microfluidics) twice at15,000 psi with cooling by ice. The lysate was centrifuged at 35,000×gfor 45 min at 4° C. The supernatant was filtered through 0.22 μm Acropak200 capsule filters (Pall).

The clarified lysate was bound to the Ni-NTA resin (Qiagen),pre-equilibrated with Ni-NTA Binding Buffer (20 mM Tris, 400 mM NaCl, 20mM Imidazole, 5 mM β-ME, pH 8.0 at 4° C.). The column was washed with500 column volumes of Ni-NTA Binding Buffer+0.1% Triton X-114 followedby 50 column volumes of the Ni-NTA Binding Buffer. The bound protein,HisRS, was eluted with 5 column volumes of Ni-NTA Elution Buffer (20 mMTris, 400 mM NaCl, 500 mM Imidazole, 5 mM β-ME, pH 8.0 at 4° C.).

The Ni-NTA eluate was further purified by an anion exchange column.Specifically, the Ni-NTA eluate was dialyzed against Q Binding Buffer(20 mM Tris, 50 mM NaCl, 1 mM DTT, pH 7.4) and loaded onto a 5 mLQ-sepharose column, pre-equilibrated with the Q Binding Buffer. Thedesired product was eluted off the column with a linear gradient of 0-1M NaCl in the Q Binding Buffer over 10 column volumes. The purifiedHisRS was concentrated and buffer exchanged into PBS (Invitrogen product#10010)+1 mM DTT, and filtered through a 0.22 μm sterile filter.

Example 3 Active Site Titration of the Cysteine Residues in Full LengthHars

To determine the location and identity of the surface exposed cysteineresidues in full length HARS, purified recombinant protein was incubatedwith iodoacetamide under native and denatured conditions to alkylate anysurface exposed cysteine residues. Samples were then analyzed bylimiting proteolysis followed by LC-mass analysis to determine thelocation and identity of the modified cysteine residues.

To perform the alkylation studies, full length, polyhistidine taggedHARS (6.65 mg/ml in PBS, 10% glycerol, 2 mM DTT, pH7.4, (Example 2) wasfirst fully reduced by incubation with 10 mM DTT for 45 minutes at roomtemperature. Incubations with iodoacetamide were conducted with aniodoacetamide concentration at either 30 mM (“Low”) or a 100 mM (“High”)for 30 minutes in the dark, and were conducted on native and denaturedsamples of HARS to confirm that the reaction was successful. DenaturedHARS was prepared by pre-incubation of the protein with 4M guanidine for45 min at 50 C. After incubation with iodoacetamide, samples weredialyzed in PBS pH 7.4 at 4 C using 10 KDa molecular weight cutoffdialysis membrane, and with at least 3 buffer exchanges, and then usedfor mass spectroscopy analysis as described below.

In brief, samples were prepared by diluting the proteins into 0.1%formic acid to a final concentration of 1 m/ml and 5 μg samples of theproteins were injected and analyzed by reverse phase HPLC followed bymass spectrum analysis using an Agilent TOF mass spectrometer. Sampleswere first separated on a C3 HPLC column (Agilent ZORBAX 300SB-C3, 5 μm,2.1×150 mm column) using a linear gradient of (mobile phase B of 2-60%)over 18 min (mobile phase A: 0.1% formic acid; mobile phase B: 0.1%formic acid in acetonitrile). Mass spectrometry analysis of the sampleswas in profile mode. Data was acquired and analyzed by MassHunter(Agilent). Measured molecular weight was calculated by MassHunterBioconfirm Agilent).

The results (data not shown) demonstrated that under native conditionsonly 3 or 4 cysteine residues are readily modified, whereas bycomparison when the protein is first denatured to disrupt its nativeconformation all 10 cysteines were readily denatured.

To identify the identity of the modified cysteine residues, samplesbefore and after incubation with iodoacetamide were subjected todenaturation in 4 M Guanidine HCl at 3TC for 30 min followed byproteolytic cleavage with LysC using a by a 10:1 ratio (w/w) at roomtemperature for 20h. Protein digests were analyzed by LC/MS/MS usingDionex HPLC and Thermo LTQ XL mass spectrometer. Samples were firstseparated on C18 HPLC column (Agilent ZORBAX 300SB-C18, 5 μm, 2.1×150mm) using a gradient of mobile phase B (mobile phase A: 0.1% formicacid; mobile phase B: 0.1% formic acid in acetonitrile). The gradientstart off with 1-3% B in 10 min and then to 40% B in 76 min. Separatedprotein digests were analyzed either by full MS in profile mode or by afull MS scan were analyzed by tandem MS/MS scan on the top threeidentified ions. Data was acquired and analyzed by Xcalibur (Thermo).Peptide sequencing was based on the MS/MS spectra of each peptide, inwhich b- and γ-ion peaks match their theoretical ions. Identification ofthe peptides and mapping of the modification sites are based on themolecular weight and confirmed by peptide sequencing using MS/MSspectra, and are listed in Table E1.

TABLE E1 LC-MS Peptide mapping results after limiting trypsin digestionCys From- RT residue To Sequence (min) MH+ Cys83  76-85 VFDVIIR CFK (SEQ ID NO: 271) 56.24 1239.68 Cys174; 155- VYRRDNPAMTRGRYREFYQ CDFDIAGNFDPMIPDAE C L 61.27 4673.14 Cys191 193 K (SEQ ID NO: 272) Cys196194- IM C EILSSLQIGDFLVK (SEQ ID NO: 273) 73.14 1909.01 210 Cys224 211-VNDRRILDGMFAI C GVSDSK (SEQ ID NO: 274) 58.53 2196.08 230 Cys235 231-FRTI C SSVDK (SEQ ID NO: 275) 22.8 1155.57 240 Cys235 231- FRTI CSSVDKLDK (SEQ ID NO: 276) 28.77 1511.79 243 Cys379 377- VP CVGLSIGVERIFSIVEQRLEALEEK (SEQ ID NO: 277) 81.00 3013.63 403 Cys445 448-LLNQLQY C EEAGIPLVAIIGEQELK (SEQ ID NO: 278) 72.46 2784.48 472 Cys505;500- RRTGQPL C I C  (SEQ ID NO: 279) 27.17 1146.57 Cys509 509

The results revealed (data not shown) that Cys235, Cys507 and Cys509 arereadily modified by iodoacetamide treatment and are thus likely to besurface-exposed residues that are readily amenable to chemicalmodification.

Example 4 Creation of Modified Hrs Polypeptides with Altered CysteineContent

To determine whether any of the 10 naturally-occurring cysteine residuesin full length HRS could be mutated to alternative naturally occurringamino acid residues, or deleted, primers were designed to selectivelymutate each cysteine residue. To accomplish this, primers based on thefollowing may be used (see Table E2).

TABLE E2 SEQ ID Mutation Oligo sequence NO: C835′-GTTTGACGTAATCATCCGTTGCTTCAAGCGCCACGGTGCAG-3′ 280 (Forward) C835′-CTG CAC CGT GGC GCT TGA AGC AAC GGA TGA TTA CGT CAA AC-3′ 281(Reverse) C174 5′-GCCGATACCGGGAATTCTACCAGTGTGATTTTGACATTGCTGGG-3′ 282(Forward) C1745′-CCC AGC AAT GTC AAA ATC ACA CTG GTA GAA TTC CCG GTA TCG 283 GC-3′(Reverse) C191 5′-CCATGATCCCTGATGCAGAGTGCCTGAAGATCATGTGCGAG-3′ 284(Forward) C1915′-CTC GCA CAT GAT CTT CAG GCA CTC TGC ATC AGG GAT CAT GG-3′ 285(Reverse) C196 5′-GCAGAGTGCCTGAAGATCATGTGCGAGATCCTGAGTTCACTTC-3′ 286(Forward) C1965′-GAA GTG AAC TCA GGA TCT CGC ACA TGA TCT TCA GGC ACT CTG C-3′ 287(Reverse) C224 5′-CTAGATGGGATGTTTGCTATCTGTGGTGTTTCTGACAGCAAGTTC-3′ 288(Forward) C2245′-GAA CTT GCT GTC AGA AAC ACC ACA GAT AGC AAA CAT CCC ATC 289 TAG-3′(Reverse) C235 5′-CAGCAAGTTCCGTACCATCTGCTCCTCAGTAGACAAGCTGG-3′ 290(Forward) C2355′-CCA GCT TGT CTA CTG AGG AGC AGA TGG TAC GGA ACT TGC TG-3′ 291 C3795′-GGGCGCAAGGTGCCATGTGTGGGGCTCAGCATTGGGG-3′ 292 (Forward) C3795′-CCC CAA TGC TGA GCC CCA CAC ATG GCA CCT TGC GCC C-3′ 293 (Reverse)C455 5′-CTGAACCAGTTACAGTACTGTGAGGAGGCAGGCATCCC-3′ 294 (Forward) C4555′-GGG ATG CCT GCC TCC TCA CAG TAC TGT AAC TGG TTC AG-3′ 295 (Reverse)C507 5′-GAGAACAGGCCAGCCCCTCTGCATCTGCTAGAACCCAGC-3′ 296 (Forward) C5075′-GCT GGG TTC TAG CAG ATG CAG AGG GGC TGG CCT GTT CTC-3′ 297 (Reverse)C509 5′-CCAGCCCCTCTGCATCTGCTAGAACCCAGCTTTCTTG-3′ 298 (Forward) C5095′-CAA GAA AGC TGG GTT CTA GCA GAT GCA GAG GGG CTG G-3′ 299 (Reverse)Last 3 5′ GAACAGGCCAGCCCCTCTAGAACCCAGCTTTCTTG 3′ 300 codon (Forward)removal Last 3 5′-CAA GAA AGC TGG GTT CTA GAG GGG CTG GCC TGT TC-3′ 301codon (Reverse) removal

To confirm the active site titration data, the crystal structure of fulllength HRS was analyzed using the program Getarea1.1 to assess therelative location of the 10 cysteine residues. The results (data notshown) suggest that in addition to C235, C507 and C509, the cysteines atpositions C174, C191 and C224 of SEQ ID NO:1, are at least partiallyexposed to the surface and could likely be modified via standardreagents. Additionally, analysis of the crystal structure of HRSsuggests that C174 and C191 are capable of making an internal disulfidebond, while C507 and C509 are capable of making interchain disulfidebonds within the HRS dimer, both potentially contributing tomicroheterogeneity that could be beneficially eliminated.

To directly assess the significance of the two C-terminal cysteineresidues in contributing to interchain disulfide bond formation,His-tagged versions of the full length and the C-terminally deletedversions of HRS (HRS(1-506)) were compared by SDS PAGE analysis beforeand after reduction. The results, shown in FIG. 3, demonstrate that fulllength HRS is a 50:50 mixture of non-covalent and SS-linked dimer, whileHRS(1-506) dramatically reduces the SS-linked dimer. Comparison of thetwo proteins by competitive ELISA, as described below, revealed thatboth proteins had comparable IC50 values with respect to Jo-1 antibodybinding (data not shown). The dramatically reduced interchain disulfidebond formation associated with HRS(1-506) suggests that this variant isa suitable starting point for the development of improved nextgeneration product forms.

To determine whether any of the remaining four partially exposedcysteine residues in full length HRS could be mutated to alternativenaturally occurring amino acid residues, primers were designed toselectively mutate C174, C191, C224 and C235 residues. To accomplishthis, the following primers were used as listed in Table E3:

TABLE E3 SEQ ID Mutation Oligo sequence NO: C1CCCGGATGCCGAGGCTTTGAAAATTATGTG (Forward) 302 C191ACAC ATA ATT TTC AAA GCC TCG GCA TCC GGG (Reverse) 303 C191SGATCCCGGATGCCGAGAGTTTGAAAATTATGTGTG (Forward) 304 C191SCAC ACA TAA TTT TCA AAC TCT CGG CAT CCG GGA TC (Reverse) 305 C191VGATCCCGGATGCCGAGGTTTTGAAAATTATGTGTG (Forward) 306 C191VCAC ACA TAA TTT TCA AAA CCT CGG CAT CCG GGA TC (Reverse) 307 C174ACGCGAATTCTATCAGGCTGATTTTGATATTGCCGG (Forward) 308 C174ACCG GCA ATA TCA AAA TCA GCC TGA TAG AAT TCG CG (Reverse) 309 C174VCGCGAATTCTATCAGGTTGATTTTGATATTGCCG (Forward) 310 C174VCGG CAA TAT CAA AAT CAA CCT GAT AGA ATT CGC G (Reverse) 311 C224SGGTATGTTTGCTATTTCCGGTGTTTCTGATTCC (Forward) 312 C224SGGA ATC AGA AAC ACC GGA AAT AGC AAA CAT ACC (Reverse) 313 C235SCCAAATTCCGTACAATCTCCTCAAGCGTGGACAAATTGG (Forward) 314 C235SCCA ATT TGT CCA CGC TTG AGG AGA TTG TAC GGA ATT TGG 315 (Reverse) C191ACCCGGATGCCGAGGCTTTGAAAATTATGTG (Forward) 316 C191ACAC ATA ATT TTC AAA GCC TCG GCA TCC GGG (Reverse) 317

Mutations were introduced by mutagenesis using the QuikChange LightningSite-Directed Mutagenesis Kit (Agilent, cat. no. 210518) following themanufacturer's instructions. After mutagenesis, the sample were treatedwith Dpn I enzyme at 37° C. and transformed into XL10 gold competentcells using routine procedures. Multiple colonies are grown in terrificbroth overnight at 37° C. and the resulting plasmids are purified withQIAprep Spin Miniprep Kit (Qiagen cat. no.27106). The plasmids aresequenced to confirm the identity of the amino acid substitution of eachclone. The representative clones were transformed into NovaBluecompetent cells (Novagen cat. no. 70181) and grown in 250 ml M9YE mediumat 37° C. overnight. A maxiprep was performed using the HiSpeed PlasmidMaxi Kit (Qiagen cat. no.12663) to create a plasmid stock of mutant forfurther analysis. The concentration and purity were determined bymeasuring A260, A280 and A230. The purified plasmids were stored at −20°C. before transfection into E. coli or mammalian cells followingstandard protocols.

To assess the impact of the mutation of the mutation of each residue,representative clones were transformed into E. coli, or mammalian cells,and the production yields, endotoxin contents, stability and relativeactivity in an ELISA assay to determine Jo-1 antibody binding asdescribed below.

Protein Production.

BL21(DE3) competent cells (Novagen, cat. no. 69450) or W3110 cells(ATTC) were transformed with the codon-optimized expression constructsencoding the reduced cysteine constructs as described above. Theexpression system, fermentation media, fermentation conditions andpurification steps used to produce recombinant protein were essentiallythe same as those described in Example 4 below, after adjusting for theproduction scale, and amount of cell paste used. Table E4 below showsthe purification yields, and endotoxin levels for the proteins made.

TABLE E4 Purification yields and endotoxin levels of reduced cysteinevariants Yield Endotoxin Name (mg/g cell paste) (EU/mg) HRS(1-506) +++0.32 HRS(1-506)C174V ++ 0.71 HRS(1-506)C174A ++ 0.30 HRS(1-506)C191A ++0.46 HRS(1-506)C191V +++ 0.33 HRS(1-506)C1191S +++ 0.32 HRS(1-506)C224S++ 0.54 HRS(1-506)C235S +++ 0.60 +++ greater than 7 mg protein/g cellpaste; ++ greater than 5 mg/g cell paste + less than 5 mg/g cell paste.

The results show that all of the reduced variants were relatively wellexpressed, and were successfully purified with low endotoxin levels. Inparticular the reduced cysteine variants based on the mutation ofCys191, and Cys235 displayed favorable expression levels; though allclones exhibited reasonable levels of expression, and low endotoxinlevels.

To assess the impact of the cysteine mutations on the chargeheterogeneity of the purified proteins, samples of each clone wereanalyzed by isoelectric focusing. Samples (10 μg) were loaded onto anisolectric focusing gel (pH 3-10) using a Life Technologies Novex pH3-10 IEF gel 1.0 mm (Cat No. P/N EC6645BOX), Novex IEF Marker 3-10 (CatNo. P/N 391201), Novex pH 3-10IEF buffer kit (Cat. No. P/N LC5317), runwith 1× cathode buffer (upper chamber) and 1× anode buffer (lowerchamber) at 100V for 1 hour, 200V for 1 hour, and 500V for 30 minutes.Gels were fixed with 12% TCA with 3.5% sulfosalicylic acid for 30minutes and stained with Expedeon InstantBlue (Cat No. P/N ISB1L). Thedata, (results not shown) demonstrate that the mutation of the cysteineat position 174 significantly reduced isoelectric point heterogeneity,consistent with the possibility that this cysteine residue undergoes anintramolecular disulfide bond formation with cysteine 191.

To assess the impact of the cysteine modifications on the thermalstability, aggregation propensity, structure, and tRNA synthetaseactivity of the resultant proteins, the proteins were assessed bydifferential scanning fluorimetry, size exclusion HPLC (SE-HPLC),competitive ELISA and active site titration. The results are shown inTable E5 below.

Differential scanning fluorimetry was performed on protein samples bymonitoring fluorescence as a function of the fluorescence intensity of alipophilic dye during thermal denaturation. Studies were carried onsamples after they were diluted to 0.5 mg/mL into 1004 final volume ofPBS pH 7.0 (150 mM NaCl, 20 mM phosphate) and mixed with a thermal shiftdye solution, which was prepared by diluting the stock solution (AppliedBiosystems/Life Technologies, P/N 4461146) 20-fold in ultrapuredistilled water (Gibco, P/N 10977). Five μL of the diluted dye was addedto 100 μL of sample. The mixture was plated into a 384 well clearoptical reaction plate (Applied Biosystems/Life Technologies P/N4309849) at 20 μL each well and 4 well replicates per sample. The platewas read by the ViiA 7 Real Time PCR Instrument (Applied Biosystems/LifeTechnologies, P/N 4453552). The thermal denaturation protocol commencedwith a rate of change of 1.6° C./s, until a temperature of 25° C. wasattained, at which point the instrument held this temperature for 2minutes, before further increasing the temperature to 99° C., at a rateof 0.5° C./s at which point this temperature was held for a further 2minutes.

Size exclusion HPLC analysis was completed on the purified proteinsamples using a TSKgel Super SW3000, 4.6 mm ID×30 cm, 4 am particlesize, 250 A column (Tosoh, 18675) using a mobile phase of 200 mMNaPhosphate, 150 mM NaCl pH 7.0, at a flow rate of 0.3 ml/min, with anAgilent 1260 HPLC system equipped with a vacuum degasser,binary/quaternary pump, thermostatted autosampler, thermostatted columncompartment, diode array detector (DAD), and Chemstation chromatographysoftware). Un-diluted samples (40 μg) of each protein were injectedafter brief centrifugation. System suitability sample (bovine serumalbumin, BSA, Thermo Scientific, P/N: 23209) and internal control (wildtype HRS) were used to bracket samples to ensure the validity of thetest.

Competitive ELISAs were performed in 96-well plates (Immulon 4HBX) whichhad been coated with a 50 μL solution of full length his-tagged HARS,adjusted to a concentration of 2 μg/mL with PBS. Plates were sealed andincubated overnight at 4° C. Prior to use, plates were washed five timeswith PBST and subsequently blocked with 100 al 1% BSA in PBS for onehour at room temperature. While the ELISA plates were blocking, thereduced cysteine competition molecules (over a concentration range of1×10⁻⁶ M to 1×10⁻¹³ M) were incubated with α-Jo-1 antibodies (GenWayGWB-FB7A3D or Immunovision HJO-0100) at 1:10,000 dilution in 1% BSA PBSin a separate incubation plate (Costar 3357 96-well) for one hour at 4°C. After blocking was finished, the ELISA plates were washed three timeswith PBST and 50 μL of solution containing antibody and competitor wasadded to the ELISA plate and the samples incubated at room temperaturefor 1.5 hours. Following initial binding incubation, plates were washedfive times with PBST. Next, 50 μL of detection antibody (AbD SerotecGoat Anti-human IgG F(ab′)2:HRP 0500-0099) was added a 1:5,000 dilutionand incubated for one hour at room temperature. Following secondarybinding incubation, plates were washed with five times PBST and 50 μLTMB substrate (Thermo Scientific Pierce TMB Substrate PI-34021) wasadded. Reactions proceeded for 8 minutes at which point 50 μL of 2Msulfuric acid stop solution was added. Colorimetric quantification wasperformed using a SpectraMax plate reader at 450 nM.

To determine the number of catalytic active sites in each HARS506cysteine variant the active site titration assay (as described in Fershtet al., (1975) Biochemistry) was employed. Briefly, assays wereperformed at room temperature with 5 μM HARS, 10 mM MgCl2, 50 μM ATP, 20mM L-histidine, 2 ug/mL inorganic pyrophosphatase, 1.65 μM [γ-32P]ATP instandard buffer (100 mM HEPES pH 7.5, 20 mM KCl). Reactions wereinitiated with enzyme in low profile PCR plates and time points werequenched in 96-well PVDF multiScreen filter plates Millipore containingHClO4/charcoal slurry (1:4 7% HClO4:10% charcoal slurry) at 30s, 1 min,2 min, 4 min, 6 min and 10 min. After mixing up and down by pipettingand samples were spun into a collection plate with Supermix scintillant,and counted in a Microbetae plate reader.

TABLE E5 Effect of cysteine modification on thermal stability,aggregation and activity of HARS % Low Active molecular IC50 by siteName Tm weight aggregates ELISA Assay titration HRS(1-506) 49.0 2.0 0.1563.3 HRS(1-506)C174V 47.8 7.8 0.39 55.5 HRS(1-506)C174A 49.2 3.0 0.1959.8 HRS(1-506)C191A 44.7 5.1 0.14 66.2 HRS(1-506)C191V 47.8 1.8 0.1660.8 HRS(1-506)C191S 45.8 2.3 0.16 63.2 HRS(1-506)C224S 48.9 4.9 0.1460.5 HRS(1-506)C235S 48.8 3.1 0.14 64.6

The results from these studies confirm that all of the cysteine mutantsare active, with little or no loss in activity, stability, orconformation as measured by active site titration, ELISA binding and Tmdeterminations for thermal denaturation. Active site titration of tRNAsynthetase activity reveals that all of the reduced cysteine mutants areactive, and thus suitable for use in any of the compositions, methodsand kits of the invention. In general the Cys191 substitutions displayedoverall lower thermostability, while the Cys174 mutants exhibitedsignificantly less heterogeneity as determined by isoelectric focusing.

Example 5 Creation of Modified (Tag Free) Hrs Polypeptides with aC-Terminal Truncation (HisRS^(N8)) or (HRS(1-506)

To delete the last three amino acids and the linker between wild typeHisRS and the His-tag, primers were designed for use with QuikChangeLightning Site-Directed Mutagenesis Kit (Agilent, cat no 210519). Toaccomplish this, the following primers are used as listed in Table E6.

TABLE E6 SEQ ID Mutation Oligo sequence NO: Delete5′-CGCCGCACCGGTCAACCGTTACACCACCACCACCACCACTG-3′ 318 CICAAALE (Forward)Delete 5′-CAG TGG TGG TGG TGG TGG TGT AAC GGT TGA CCG GTG 319 CICAAALECGG CG-3′ (Reverse)

The deletion was made per the QuikChange Lightning Site-DirectedMutagenesis Kit manufacturer's instructions. After mutagenesis, thesample was treated with Dpn I enzyme at 37° C. and transformed into XL10gold competent cells using routine procedures. Multiple colonies weregrown in luria-bertani broth overnight at 37° C. and the resultingplasmids were purified with QIAprep Spin Miniprep Kit (Qiagen cat.no.27106). The plasmids were sequenced to confirm the identity of theamino acid substitution of each clone. To delete the His tag, primerswere designed for use with QuikChange Lightning Site-DirectedMutagenesis Kit (Agilent, cat no 210519). To accomplish this, thefollowing primers were used as listed in Table E7.

TABLE E7 SEQ ID Mutation Oligo sequence NO: Delete His-tag5′-CGC CGC ACC GGT CAA CCG TTA TGA GAT CCG GCT 320 Forward GCT AAC-3′Delete His-tag 5′-GTT AGC AGC CGG ATC TCA TAA CGG TTG ACC GGT 321Reverse GCG GCG-3′

The deletion was made per the QuikChange Lightning Site-DirectedMutagenesis Kit manufacturer's instructions, as described above.

Protein Production.

BL21(DE3) competent cells (Novagen, cat. no. 69450) or W3110 cells(ATTC) were transformed with the codon-optimized expression constructencoding HisRS' (HRS(1-506)) as described in Example 2. The expressionsystem, fermentation media, and fermentation conditions used to producerecombinant proteins were essentially the same as those described inExample 2.

Purification of Tag-Free HisRSN⁸ (HisRS(1-506)).

Frozen cell paste (400 g) was resuspended in 4-volumes (1600 mL) ofLysis Buffer (50 mM Tris, 50 mM NaCl, 5 mM MgCl₂, 2 mM L-Cysteine,pH7.4). Complete EDTA-FREE protease inhibitor tablets (Roche, Cat #05056 489 001) were added to the suspension at a ratio of 1 tablet/50 mL.The suspension was passed through a microfluidizer (Microfluidics) twiceat 18,000 psi with cooling by ice. The lysate was centrifuged at15,000×g for 45 min at 4° C. The supernatant was filtered through 2-3AcroPak 1500 capsules (0.8/0.2 μm, Pall, PN12675).

The clarified lysate was loaded onto a 382 ml Q HP column (5×19.5 cm)pre-equilibrated with Q Buffer A (50 mM Tris, 50 mM NaCl, pH 7.4). Theproduct was eluted with a linear gradient of 0-30% Q Buffer B (50 mMTris, 1 M NaCl, pH 7.4) over 10 column volumes (CV). Fractions werecollected at ½ CV/fraction and analyzed by SDS-PAGE. Pooling was basedon gel analysis.

A 3.5 M ammonium sulfate solution was added to the Q HP pool above to afinal concentration of 1.2 M. The mixture was filter through an AcroPak200 (0.2 um) and loaded onto a 481 ml Phenyl HP column (5×24.5 cm)pre-equilibrated with 20 mM Tris, 1.2 M ammonium sulfate, pH 7.0. Theproduct was eluted with a linear gradient of 1.2-0 M ammonium sulfate in20 mM Tris/pH 7.0 over 10 CV. Fractions (½ CV/fraction) containing theproduct based on SDS-PAGE analysis were pooled.

The Phenyl Pool from above was concentrated to 0.5 L via a TFF system,consisting of a Pellicon Mini cassette holder (Millipore Cat#XX42PMINI),a Masterflex I/P pump, and 2×0.1 m² cassette (30 kD MWCO, NovasepCat#PP030M01L). The concentrated solution was then buffer exchanged with6 diavolumes (3 L) of CHT Buffer A (10 mM sodium phosphate, 150 mM NaCl,pH 7.0). The retentate was filtered through a 0.2 μm Millex GP-50 filter(Millipore part #SLGP 05010) before proceeding to the next step.

The above solution was loaded onto a 380 ml ceramic hydroxyapatite (CHT)column (5×19.4 cm) pre-equilibrated with CHT Buffer A. The column waswashed with Buffer A and followed by 6% Buffer B (500 mM sodiumphosphate, 150 mM NaCl, pH 7.0). The product was eluted with a lineargradient of 6-56% Buffer B over 10 CV. Fractions (½ CV/fraction)containing the product based on SDS-PAGE analysis were pooled.

Using the same TFF system, the CHT Pool was concentrated to −0.2 L,buffer exchanged with 6 diavolumes of the current formulation buffer (20mM sodium phosphate, 150 mM NaCl, pH 7.0), and concentrated to a targetconcentration of −10 mg/ml. The product solution was filtered through a0.2 μm Millex GP-50 filter (Millipore part # SLGP 05010), and stored in−80° C. freezer.

Example 6 Evaluation of HRS(1-60) (Resokine) as an Anti-InflammatoryAgent

To evaluate the potential anti-inflammatory property of HRS derivedpolypeptides, an N-terminal, naturally occurring splice variantcomprising amino acids 1-60 of HRS (“Resokine”) was tested in a TNBSinduced model of colitis. Studies were performed in male BDF-1 mice,with 12 mice/group; Budesonide was added at 5 mg/kg orally.

In this study Resokine was administered daily by IV injection, starting3 days prior to TNBS treatment, at a concentration of 1 or 5 mg/Kg. Thedata shown in FIG. 4 demonstrates that treatment with Resokine at eitherconcentration resulted in a significant increase in survival.Accordingly Resokine appears to have potent anti-inflammatory effects,consistent with the hypothesis that HRS polypeptides are involved in thelocal control of inflammatory processes.

Example 7 Evaluation of Hrs Polypeptides for the Treatment ofStatin-Induced Myositis and Rhabdomyolysis

Statins are HMG CoA Reductase inhibitors which inhibit the synthesis ofmevalonate, the rate limiting step in cholesterol synthesis. Statintherapy has proved beneficial in lowering cholesterol levels inpatients. However, side-effects and complications of statin therapyinclude muscle weakness, myositis and rhabdomyolysis. Muscle myopathy isa complication with several statins on the market and patients are oftenremoved from their statin-therapy if they exhibit any of these symptoms.Like many other myopathies, muscular dystrophies and inflammatorydisorders of muscle, disease progression in statin induced myopathyappears to occur as the result of an initial chemical, genetic orphysical injury, which becomes increasingly inflamed as a result ofimmune cell invasion into the damaged muscle cells.

Accordingly statin induced myopathy represents a broadly applicablemodel system to study drug induced myositis, which is directlyapplicable to other myopathies and muscular dystrophies, all of whichall share a common inflammatory component which mediates diseaseprogression by promoting immune cell invasion of the damaged muscletissue.

The purpose of this study was to evaluate the efficacy of HRS(1-506) inreversing the effects of statin-induced muscular myositis, as indicatedby altered circulating enzyme levels, and changes in gene expression ofmuscle function and inflammatory markers in response to treatment withHRS(1-506).

To achieve this, rats were dosed daily with 1 mg/kg Cerivastatin andthen switched to an every other day (qod) dosing with Cerivastatin. Thegoal of this dosing regimen was to maintain a sustained disease state inthe animals, but not to have such severe disease that rat survival isgreatly impacted. The efficacy of a dose range of HRS(1-506) was thenevaluated in rats after statin-dosing had already initiated measurablechanges in circulating markers of myositis.

Protocol and Methods.

In this study, 10 week old female Sprague-Dawley rats were treated with1 mg/kg Cerivastatin ((Sigma, Cat No. SML0005) in 0.5% methylcellulose,starting on day 1 via oral gavage. After 7 days of daily administration,rats were switched to an every other day dosing strategy (qod) on days9, 11 and 13. HRS(1-506) and vehicle administration were initiated onday 6 through intravenous injection and rats were dosed daily to day 14(shown schematically in FIG. 5A). All rats were taken down on day 15, 24hours after the final test article dosing and 48 hours after the laststatin administration. HRS(1-506) was administered at 3 doses (0.3, 1.0and 3.0 mg/kg) in 20 mM NaPO4, 0.15M NaCl, pH 7.0 daily.

To address the primary objective of this study, the following studymeasurements and endpoints were performed: rat survival, weight,circulating serum CK levels at days 12 and 15, H&E staining on day 15hamstring samples, Troponin-I ELISA, CBC on day 15 blood, qPCR onhamstring samples and serum endogenous HARS levels.

qPCR methods.

Mouse hamstring was excised from the animals and stored at −80° C. untilanalysis. Tissue was prepped in groups of 10 hamstrings using Qiagen'sRNeasy Fibrous Tissue Midi Kit (Catalog #75742). Once RNA was elutedfrom the Qiagen column, it was run on an Agilent's Bioanalyzer 2100 totest RNA integrity and NanoDrop to determine RNA concentration andpurity. RNA was then stored at −80° C.

Reverse transcription (RT) of RNA to cDNA was performed in a 96 well PCRplate format in Eppendorf's Mastercycler PCR machine with the followingprogram: 37° C. for 60 minutes, 95° C. for 5 minutes. The edge wells ofthe 96 well plate were not used and filled with 50 mcL water to preventevaporation of inside wells. 20 mcL RNA and 30 mcL of reversetranscription master mix (Ambion's TaqMan PreAmp Cells to CT Kit catalog#4387299) was used per sample RT. Once RT was completed, next step wasto pre-amplify genes of interest in the sample cDNA. Primers of genes ofinterest (DELTAgene primers designed by Fluidigm) were combined to afinal concentration of 200 nM. Using these primers, genes of interestwere pre-amplified in each sample. Pre-amplification was performed in 10mcL reactions (2.5 mcL cDNA, 7.5 mcL Pre-Amp mastermix) in 384-wellformat using an Applied Biosystems ViiA7 PCR machine with the followingprogram: 95° C. for 10 minutes, 14 cycles of 95° C. for 15 seconds and60° C. for 4 minutes. After pre-amplification step, exonuclease (NewEngland BioLabs catalog #M0293L) was added to remove unincorporatedprimers from each sample. This exonuclease reaction was also completedin the ViiA7 PCR machine with the following program: 37° C. for 30minutes, 80° C. for 15 minutes. After exonuclease, the RT sample wasfurther diluted 1:5 (7 mcL exonuclease sample+18 mcL low EDTA buffer).

The chip used to run qPCR on Fluidigm's Biomark system was a 96.96Dynamic Array IFC for Gene Expression. The chip was first primed withthe IFC controller HX as per manufacturer's recommendations beforesample and assays were loaded. To prepare assays to be loaded on a chip,4.4 mcL assay master mix (Fluidigm's 2× Assay Loading Reagent catalog#8500736 and low EDTA TE) to 3.6 mcL 20 mcM forward and reverse primersfor each gene of interest were prepared in a 96 well plate. To preparesamples, 4.5 mcL sample master mix (Ambion's 2× TaqMan Gene ExpressionMaster Mix, Fluidigm's 20×DNA Binding Dye Sample Loading Reagent catalognumber 100-0388, and Biotium's 20× EvaGreen catalog #31000) was added to3 mcL diluted pre-amplified/exonuclease sample in a 96 well plate. Oncethe chip had been primed, 5 mcL sample or assay prepared above wereloaded onto the chip. The chip was them returned to the IFC controllerfor the samples to be loaded into the chip. After the chip had finishedloading, qPCR could then be run on the Biomark using preset program for96.96 Dynamic Array for Gene Expression with a melt curve to determineprimer specificity. Relative gene expression was determined by the deltadelta Ct method.

Quantification of Extracellular HARS.

A 96 well based ELISA was developed in-house using 2 mouse anti-HARSmonoclonal antibodies M03 (Sigma #SAB1403905, and Abnova #H00003035-M03)and M01 (Abgent #AT2317a) in a sandwich format to detect HARS in ratserum. Assays were run in 96 well Costar plates (Costar 96-well plate#3369) using a seven point standard curve which was generated rangingfrom 75 to 0.1 ng/ml using a stock solution of HRS(1-506); (7.5 mg/ml in20 mM NaPO4, 0.15 M NaCl pH 7.0, using 1x PBST (0.05% Tween-20) as adiluent). The M01 mouse monoclonal, clone 1C8 (Abgent #AT2317a) wasbiotinylated in house and used as the detection antibody, and the M03mouse monoclonal antibody (Sigma #SAB1403905, lot#11238, 0.5 mg/mL andAbnova #H00003035-M03, lot#11238, 0.5 mg/mL) was used as a captureantibody. Casein (Thermo Scientific #37528) was used as a blockingagent, and 1x PBST (0.05% Tween-20) was used as a wash buffer. Antibodybinding was quantified using Streptavidin-HRP (Invitrogen cat#434323,Lot#816755A) using TMB Substrate (Thermo #34021) and with 2M sulfuricacid as the stop solution.

ELISA assays were run by coating plates overnight with 0.6 to 2 μg/mlM03 antibody in 1×PBS, which were then blocked by incubation with caseinfor one hour 1 hour, and washed 3× with PBST. Plates were then incubatedwith standards and samples for 1 hour, washed 3×PBST, and then incubatedwith 500 ng/ml biotinylated-M01 diluted in PBST, 1 hour, washed 3×PBST,incubated with 200 ng/ml streptavidin-HRP for 1 hour, washed 3× withPBST, and then the TMB substrate added for 4 minutes. Reactions werestopped with stop solution and absorbance read at 450 nm.

The results were quantified based on the standard curve based on theaverage raw absorbance values without background subtraction. Prism wasused for standard curve fitting. Model: Log(agonist) vs. response fit[4-parameter logistic regression] Percent recovery was calculated foreach individual concentration point (not averaged) by:

$\frac{\left( {{measured} - {actual}} \right) \times 100\%}{({actual})}$

Other Readouts.

Rats were weighed daily. Serum samples were taken on days 1, 8, 12 (viatail vein) and day 15 (terminal) to be used for circulating enzymeanalysis (Idexx) and serum skeletal muscle Troponin-I measurements, weremeasured using a commercial ELISA kit. Urinalysis was performed on days3, 5, 8, 10, 12 and 15 prior to dosing on that day. CBC analysis was runon blood isolated on day 15 prior to euthanizing rats. On day 15, therats were euthanized and a portion of the hamstring muscle and lung (notinflated) was placed in 10% NBF for paraffin embedding and H&E stainingof sections (Premier Laboratory). Another portion of hamstring muscleand lung was placed at -80C to use for RNA extraction and profiling.Liver, kidney and heart were also isolated on day 15 and placed inzinc-formalin for paraffin embedding (TSRI Histology) for long-termtissue storage.

Results.

There was 100% survival in this study, and all rats survived to thescheduled takedown on day 15. Statin-dosed rats had lower averageweights than control rats not dosed with statin. On day 15, thestatin+vehicle group had the lowest average rat weight of all thegroups, whereas the Statin+3 mg/kg HRS(1-506)-dosed group had thehighest weight average of all the statin-treated animals (data notshown). CBC analysis showed overall similar patterns of changes betweendifferent animal treatment groups (data not shown).

A small increase in serum CK was observed in statin treated rats overuntreated controls on days 12 and 15. On day 12, rats dosed with 1 mg/kgand 3 mg/kg HRS(1-506) had smaller, tighter CK averages compared toStatin+Vehicle treated animals (FIGS. 6A-B), consistent with a positiveimpact of HRS(1-506) treatment on statin induced myositis, alsoconsistent with a positive effect of HRS(1-506) on muscle function,muscle troponin C levels were also reduced in HRS(1-506) treated animals(FIG. 5B). Moreover endogenous serum HRS levels were elevated instatin-treated rats compared to rats not receiving statin (FIG. 7),suggesting that the release of HRS may play a role as an endogenousregulator of muscle inflammation. H&E staining on hamstringsdemonstrated reduced muscle degeneration/necrosis and inflammationscores in statin-treated rats dosed with 1 mg/kg and 3 mg/kg HRS(1-506)compared to vehicle-dosed and 0.3 mg/kg HRS(1-506)-dosed rats (FIG. 8).

To further investigate the mechanistic basis for the effects of HRS onstatin induced myopathy, changes in gene expression in the hamstringsfrom treated animals was examined after the completion of the study. RNAprofiling was performed on hamstring muscles isolated from the rats onday 15 as described above. The results from these studies demonstratedthat all 13 genes that were elevated by more than 5 fold in response tostatin treatment were reduced by treatment with HRS(1-506) (see TableE8; and FIGS. 9-10)

TABLE E8 Gene regulated Gene regulated Gene regulated by more by more bymore than 25 fold than 10 fold than 4 fold No Change CD8a MCP1 CD11aHARS MMP9 CD8b CD11b HARS2 IL6 CCR5 CD45 DARS IL10 CD18 SDC1 GARS IFNgQARS

Transcriptional profiling of statin treated rat hamstrings: revealedthat 10 diabetes/metabolic syndrome related genes (FIG. 11) and severalhousekeeping genes (data not shown) were not significantly impacted byHRS treatment. By contrast, transcriptional profiling of statin treatedrat hamstrings of 26 immune cell marker genes revealed significantchanges in a larger number of genes (see FIGS. 12-14), including thedose dependent inhibition of ITGAL(CD11a), CD11b, CD8a, CD8b, CD18,CCR5, PTPPC and (CD45R) expression. Additionally HRS(1-506) waseffective in reducing the expression of a number of inflammatory markergenes including IL6, MCP1, IL10 and IFN gamma (see FIGS. 15-16).Transcriptional changes were also observed in 14 adhesion, development,and fibrosis related genes (see FIGS. 17-18), the muscle contractilitygene Neb (data not shown), and in genes associated with muscularwasting, atrophy, and myogenesis (see FIGS. 19-20).

Conclusions.

Decreased CK, serum Troponin-I and muscle cell degeneration/necrosis andmuscle inflammation were all observed in animals receiving higher dosesof HRS(1-506), either at 1.0 mg/kg or 3.0 mg/kg in contrast to animalsreceiving either Vehicle or low dose 0.3 mg/kg HRS(1-506). RNA profilingdata supported these results by demonstrating reduced CD8a, IL-6, MCP-1and MMP-9 expression in hamstrings of statin-treated rats dosed withhigher doses of HRS(1-506). Up-regulation of these genes is most likelydue to increased immune cell infiltrate into damaged muscle tissue.Based on the identity of the expressed genes, the infiltrating immunecells are likely to be made up of one of more of the following celltypes, T cells, dendritic cell, NK cells, and macrophage/monocytes. Allof these cell types have been associated with muscle inflammation, andthe ability of the HRS polypeptides, including HRS(1-506) to mediate adramatic inhibition of this immune cell influx suggests that HRSpolypeptides such as HRS(1-506) represent potent immunoregulators, whichare capable of acting as potent immunomodulators in a broad range ofinflammatory and autoimmune diseases and disorders.

Example 8 Preparation of HRS-Fc Polypeptides

N-terminal and C-terminal Fc-histidyl tRNA synthetase (HRS-Fc) fusionproteins were prepared, purified, and analyzed as follows.

Plasmid Construction.

The human IgG1 Fc domain was amplified by polymerase chain reaction(PCR) before inserting into the C-term or N-term of the HRS polypeptideHRS(1-60) via sequential PCR reactions using the primers below, and theresulting amplified DNA fragments inserted into C-term or N-term ofHRS(1-60) located in the pET28 expression vector (Novagen 69864). Itwill be appreciated that the creation of the N-terminal Fc fusionprotein results in the deletion/replacement of the N-terminal methioninein HRS(1-60) with the C-terminal amino acid of the Fc domain, and viceversa where appropriate.

The following primers were used to create the N-terminally fusedHRS(1-60) Fc fusion protein (Fc-HRS(2-60)) (Table E9):

TABLE E9 Primer Sequences SEQ ID Primer Name Sequence NO: FcNSV9-FTAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCTGACAAAACTC 323 ACACATGCCCFcNSV9tail AGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGG 324 FcNSV9-RCCAGCGCCGCACGCTCTGCTTTACCCGGAGACAGGGAGAGGCT 325 FcNSV9-F2TTTTGTTTAACTTTAAGAAGGAGATATACATATGTCTGACAAAACTCACA 326 CATGCCCFcNSV9tail2 CTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGC 327 FcNSV9-R2GCGCCGCACGCTCTGCTTTACCCGGAGACAGGGAGAG 328

The following primers were used to create the C-terminally fusedHRS(1-60) Fc fusion protein (HRS(1-60)-Fc) (Table E10).

TABLE E10 Primer Sequences SEQ ID Primer Name Sequence NO: FcCSV9-FCAAACAGAAATTTGTGCTCAAAACCCCCAAGTCTGACAAAACTCACAC 329 ATGCCCACCGFcC5V9tail AGCCTCTCCCTGTCTCCGGGTAAATGAGATCCGGCTGCTAACAAAGCC 330 CFcCSV9-R GGGCTTTGTTAGCAGCCGGATCTCATTTACCCGGAGACAGGGAGAGGCT 331 FcCSV9-F2CAGAAATTTGTGCTCAAAACCCCCAAGTCTGACAAAACTCACACATGC 332 CC FcCSV9tail2CTCTCCCTGTCTCCGGGTAAATGAGATCCGGCTGCTAACAAAG 333 FcCSV9-R2CTTTGTTAGCAGCCGGATCTCATTTACCCGGAGACAGGGAGAG 334

The PCR reactions were performed using recommended thermal cyclingparameters, and the PCR-amplified fragments were verified on by gelelectrophoresis. Sequences were confirmed by performing alignments withthe theoretical sequences using EMBOSS Pairwise Alignment Algorithms.The cloned DNA and protein sequences of Fc-HRS(2-60) and HRS(1-60)-Fcare shown below.

DNA sequence of Fc-HRS(2-60) (N-terminal Fc fusion): (SEQ ID NO: 335)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGC TCAAAACCCCCAAG TGADNA sequence of HRS(1-60)-Fc (C-terminal Fc fusion). (SEQ ID NO: 336)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTC TCC CTG TCT CCG GGTAAATGAProtein sequence of Fc-HRS(2-60) (N-terminal Fc fusion) (SEQ ID NO: 337)MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKProtein sequence of HRS(1-60)-Fc (C-terminal Fc fusion) (SEQ ID NO: 338)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Additional N-terminal and C-terminal Fc-histidyl tRNA synthetase(HRS-Fc) DNA constructs were prepared as follows.

Plasmid Construction.

HRS (2-60) with an N-terminal Fc or HRS (1-60) with a C-terminal Fc(Example 8) were subcloned into a modified pET24b vector (EMD,Gibbstown, N.J.) containing a TAC promoter instead of T7 (“pET24b_TAC”).The Fc-HRS (2-60) and HRS (1-60)-Fc were amplified by polymerase chainreaction (PCR) using the primers below which contain a 5′ NdeI site anda 3′ XhoI site, and the resulting amplified DNA was subcloned intopET24b_TAC using the NdeI and XhoI restriction sites.

The following primers were used to amplify the Fc-HRS (2-60) (N-terminalFc fusion) (Table E11):

TABLE E11 Primer Sequences SEQ Primer ID Name Sequence NO: 1921-NdeI-GATATACATATGTCTGACAAAACTCACACATGCC 343 FWD 1921-XhoI-GATCCTCGAGTCACTTGGGGGTTTTG 344 REV

The following primers were used to amplify the HRS (1-60)-Fc (C-terminalFc fusion) (Table E12).

TABLE E12 Primer Sequences SEQ Primer ID Name Sequence NO: 1922-NdeI-GATATACATATGGCAGAGCGTGCGG 345 FWD 1922-XhoI- GATCCTCGAGTCATTTACCCGGAGAC346 REV

The PCR reactions were performed using recommended thermal cyclingparameters, and the PCR-amplified fragments were verified by gelelectrophoresis. Sequences were confirmed by performing alignments withthe theoretical sequences using DNASTAR Lasergene SeqMan Pro. The clonedDNA and protein sequences of Fc-HRS(2-60) and HRS(1-60)-Fc are shownbelow.

DNA sequence of Fc-HRS(2-60) (N-terminal Fc fusion): (SEQ ID NO: 347)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGC TCAAAACCCCCAAG tgaDNA sequence of HRS(1-60)-Fc (C-terminal Fc fusion): (SEQ ID NO: 348)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA tgaProtein sequence of Fc-HRS(2-60) (N-terminal Fc fusion):(SEQ ID NO: 349) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPK.Protein sequence of HRS(1-60)-Fc (C-terminal Fc fusion):(SEQ ID NO: 350) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Additional N-terminal and C-terminal Fc-histidyl tRNA synthetase(HRS-Fc) DNA constructs were prepared as follows.

Plasmid Construction.

HRS (2-40), (2-45), (2-50), (2-55), (2-66) with an N-terminal Fc or HRS(1-40), (1-45), (1-50), (1-55), (1-66) with a C-terminal Fc weregenerated using Quikchange Mutagenesis (Agilent, Santa Clara, Calif.).Previously generated pET24b_TAC constructs containing Fc-HRS(2-60) andHRS(1-60)-Fc in combination with the primers listed below, were used inthe Quikchange reaction to generate the HRS-Fc constructs.

The following primers were used to amplify the Fc-HRS (2-40), (2-45),(2-50), (2-55), (2-66) polypeptides (N-terminal Fc fusion) (Table E13):

TABLE E13 Primer Sequences SEQ ID Primer Name Sequence NO:Fc-H-aa2-40 FWD 5′-GGT GGC GAA ACT CCT GAA ATG ACT CGA GGA TCC GGC TGC-351 3′ Fc-H-aa2-40 REV5′-GCA GCC GGA TCC TCG AGT CAT TTC AGG AGT TTC GCC ACC- 352 3′Fc-H-aa2-45 FWD 5′-CTG AAG GCA CAG CTG TGA CTC GAG GAT CCG GCT GC-3′ 353Fc-H-aa2-45 REV 5′-GCA GCC GGA TCC TCG AGT CAC AGC TGT GCC TTC AG-3′ 354Fc-H-aa2-50 FWD 5′-GGG TCC TGA TGA AAG CTG ACT CGA GGA TCC GGC TGC-3′355 Fc-H-aa2-50 REV5′-GCA GCC GGA TCC TCG AGT CAG CTT TCA TCA GGA CCC-3′ 356Fc-H-aa2-55 FWD 5′-GCA AAC AGA AAT TTG TGT GAC TCG AGG ATC CGG CTG C-3′357 Fc-H-aa2-55 REV5′-GCA GCC GGA TCC TCG AGT CAC ACA AAT TTC TGT TTG C-3′ 358Fc-H-add-aa61-66 5′-GCT CAA AAC CCC CAA GGG AAC CCG TGA TTA TAG TTG ACT359 FWD CGA GGA TCC GG-3′ Fc-H-add-aa61-665′-CCG GAT CCT CGA GTC AAC TAT AAT CAC GGG TTC CCT TGG 360 REVGGG TTT TGA GC-3′

The following primers were used to amplify the HRS (1-40), (1-45),(1-50), (1-55), (1-66) -Fc (C-terminal Fc fusion): (Table E14).

TABLE E14 Primer Sequences SEQ ID Primer Name Sequence NO:H-aa1-40-Fc FWD5′-GGT GGC GAA ACT CCT GAA ATC TGA CAA AAC TCA CAC ATG C- 361 3′H-aa1-40-Fc REV5′-GCA TGT GTG AGT TTT GTC AGA TTT CAG GAG TTT CGC CAC C- 362 3′H-aa1-45-Fc 5′-CTG AAA CTG AAG GCA CAG CTG TCT GAC AAA ACT CAC ACA 363FWD TGC-3′ H-aa1-45-Fc REV5′-GCA TGT GTG AGT TTT GTC AGA CAG CTG TGC CTT CAG TTT 364 CAG-3′H-aa1-50-Fc 5′-GCT GGG TCC TGA TGA AAG CTC TGA CAA AAC TCA CAC ATG C-365 FWD 3′ H-aa1-50-Fc REV5′-GCA TGT GTG AGT TTT GTC AGA GCT TTC ATC AGG ACC CAG C- 366 3′H-aa1-55-Fc 5′-GAA AGC AAA CAG AAA TTT GTG TCT GAC AAA ACT CAC ACA 367FWD TGC-3′ H-aa1-55-Fc REV5′-GCA TGT GTG AGT TTT GTC AGA CAC AAA TTT CTG TTT GCT 368 TTC-3′H-add-aa61-66-Fc 5′-GCT CAA AAC CCC CAA GGG AAC CCG TGA TTA TAG TTC TGA369 FWD CAA AAC TCA C-3′ H-add-aa61-66-Fc5′-GTG AGT TTT GTC AGA ACT ATA ATC ACG GGT TCC CTT GGG 370 REVGGT TTT GAG C-3′

The PCR reactions were performed using manufacturer recommended thermalcycling parameters. Sequences were confirmed by performing alignmentswith the theoretical sequences using DNASTAR Lasergene SeqMan Pro. Thecloned DNA and protein sequences of the HRS-Fc constructs are shownbelow.

DNA sequence of Fc-HRS(2-40) (N-terminal Fc fusion): (SEQ ID NO: 371)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCT GAAATGADNA sequence of Fc-HRS(2-45) (N-terminal Fc fusion): (SEQ ID NO: 372)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGTGA DNA sequence of Fc-HRS(2-50) (N-terminal Fcfusion): (SEQ ID NO: 373)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCTGADNA sequence of Fc-HRS(2-55) (N-terminal Fc fusion): (SEQ ID NO: 374)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGT GADNA sequence of Fc-HRS(2-66) (N-terminal Fc fusion): (SEQ ID NO: 375)ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGGGAACCCGTGATTATAGTTGADNA sequence of HRS(1-40)-Fc (C-terminal Fc fusion): (SEQ ID NO: 376)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATGADNA sequence of HRS(1-45)-Fc (C-terminal Fc fusion): (SEQ ID NO: 377)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA DNA sequence of HRS(1-50)-Fc (C-terminal Fcfusion): (SEQ ID NO: 378)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGADNA sequence of HRS(1-55)-Fc (C-terminal Fc fusion): (SEQ ID NO: 379)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT GADNA sequence of HRS(1-66)-Fc (C-terminal Fc fusion): (SEQ ID NO: 380)ATGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGGGAACCCGTGATTATAGTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAProtein sequence of Fc-HRS(2-40) (N-terminal Fc fusion):(SEQ ID NO: 381) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLK QQKASAELIEEEVAKLLKProtein sequence of Fc-HRS(2-45) (N-terminal Fc fusion):(SEQ ID NO: 382) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQL Protein sequence of Fc-HRS(2-50) (N-terminal Fcfusion): (SEQ ID NO: 383)MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESProtein sequence of Fc-HRS(2-55) (N-terminal Fc fusion):(SEQ ID NO: 384) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVProtein sequence of Fc-HRS(2-66) (N-terminal Fc fusion):(SEQ ID NO: 385) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSProtein sequence of HRS(1-40)-Fc (C-terminal Fc fusion):(SEQ ID NO: 386) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKProtein sequence of HRS(1-45)-Fc (C-terminal Fc fusion):(SEQ ID NO: 387) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Protein sequence of HRS(1-50)-Fc (C-terminal Fcfusion): (SEQ ID NO: 388)MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKProtein sequence of HRS(1-55)-Fc (C-terminal Fc fusion):(SEQ ID NO: 389) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKProtein sequence of HRS(1-66)-Fc (C-terminal Fc fusion):(SEQ ID NO: 390) MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N-terminal Fc-histidyl tRNA synthetase (HRS-Fc) tandem fusion DNAconstruct was prepared as follows.

Plasmid Construction.

HRS (2-60)_HRS (2-60) with an N-terminal Fc was generated usingpET24b_TAC_Fc-HRS (2-60) construct. The HRS (2-60) gene was PCRamplified with the primers listed below, which contain a 5′ and 3′ XhoIsite. The PCR reactions were performed using recommended thermal cyclingparameters. pET24b_TAC_Fc-HRS (2-60) construct was digested with XhoI,dephosphorylated, and gel purified. The PCR generated fragment was alsodigested with XhoI and gel purified. The gel purified HRS (2-60) wassubcloned into the XhoI site of pET24b_TAC_Fc-HRS (2-60). To generatethe final construct, QuikChange mutagenesis was used to remove the stopcodon and XhoI site between the tandem HRS (2-60) fragments using theprimers listed below. Sequences were confirmed by performing alignmentswith the theoretical sequences using DNASTAR Lasergene SeqMan Pro.

The following primers were used to amplify the Fc-HRS (2-60) (TableE15):

TABLE E15 Primer Sequences SEQ ID Primer Name Sequence NO: XhoI-H-aa2-5′-TAT TCT CGA GGC AGA GCG TGC GGC-3′ 391 60 FWD H-aa2-60-5′-GCGCCTCGAGTCACTTGGGGGTTTTG-3′ 392 stop-XhoI REV delete stop-5′-GTG CTC AAA ACC CCC AAG GCA GAG CGT GCG GCG 393 XhoI CTG G-3concatemer FWD delete stop-5′-CCA GCG CCG CAC GCT CTG CCT TGG GGG TTT TGA 394 XhoI GCA C-3′concatemer REV

DNA sequence of Fc-HRS(2-60) HRS(2-60) (N-terminal Fc fusion):(SEQ ID NO: 395) ATGTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAGGCAGAGCGTGCGGCGCTGGAGGAGCTGGTGAAACTTCAGGGAGAGCGCGTGCGAGGCCTCAAGCAGCAGAAGGCCAGCGCCGAGCTGATCGAGGAGGAGGTGGCGAAACTCCTGAAACTGAAGGCACAGCTGGGTCCTGATGAAAGCAAACAGAAATTTGTGCTCAAAACCCCCAAG TGAProtein sequence of Fc-HRS(2-60) HRS(2-60) (N- terminal Fc fusion):(SEQ ID NO: 396) MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPK

Preparation and purification of HRS(1-60)-Fc and Fc-HRS(2-60) fusionproteins. E. coli strain. The E. coli BL21-CodonPlus® (DE3) RIPLCompetent Cells (Agilent 230280) transformed with the pET expressionconstructs described above were used for initial production of Fc fusionproteins.

Media. M9YE medium was prepared by mixing sterile 5× M9 minimal salt (BD248510), yeast extract solution in sterile purified water (BD 212750),sterilized 20% glucose (Sigma G7021), and sterile 1.0 M MgSO4 (SigmaM7506). For the feeding solution, the yeast extract solution (5%),glucose solution (50%), and 10 ml concentrated trace element solution(containing Fe³⁺, Mn²⁺, boric acid, Mo⁶⁺, Co²⁺, Cu²⁺, Zn²⁺ and EDTA), aswell 10 ml magnesium sulfate solution, were autoclaved separately. Thecomponents were mixed just prior to the fed-batch phase. Kanamycinsulfate was added to a final concentration of 100 μg/ml in the culturemedium.

Fed-batch fermentation. A 0.5 L Multifors fermentors (HT-Infors) withIris software was used for the fed-batch fermentation process. Theagitation was set at 1000 rpm. The pH value was controlled at 7.0automatically by the addition of 30% ammonium hydroxide (Sigma 221228)and 30% phosphoric acid (Sigma P5811). Air was provided at a flow rateof 0.5 L/min with an oil-free diaphragm air compressor (Cole-Parmer) andpassed through a 0.2 μm filter. The dissolved oxygen level wascontrolled at 70% by providing pure oxygen (West Air). The temperaturewas controlled at 30° C. with a Neslab RTE7 circulator (ThermoScientific). Foaming was controlled by addition of the antifoam 204(Sigma A8311).

The initial volume of M9YE medium in the fermentor was 0.3 L. Thefermentor was inoculated with 15 ml of the seed culture grown overnightat 30° C. and 250 rpm. When the carbon source was depleted in thevessel, the concentrated feeding solution was introduced into the vesselby a peristaltic pump at 0.12 ml/min. When the optical density of thecells at 600 nm reached exponential phase, the culture was induced with0.5 mM IPTG (Fisher Scientific BP1755). The culture was grown overnight(about 17-hour induction) and the final OD₆₀₀ reached about 120. Thecells were harvested by centrifugation at 8,000 g for 30 min. Thesupernatant was decanted and the pellet was stored at −20° C. untilpurification.

Additional HRS-Fc fusion proteins were prepared using a modified pET24bvector (EMD, Gibbstown, N.J.) containing a TAC promoter instead of T7(“pET24b_TAC”) and transformed into UT5600 competent cells. UT5600competent cells were prepared from bacterial stock obtained from theColi Genetic Stock Center (CGSC, Yale). UT5600 is a K12 derivativestrain of E. coli and is designated as genotype: F, araC14, leuB6(Am),secA206(aziR), lacY1, proC14, tsx-67, A(ompT-fepC)266, entA403,glnX44(AS), trpE38, rpsL109(strR), xylA5, mtl-1, thiE1.

Expression vectors comprising these constructs were transformed intoUT5600 cells using standard procedures, and glycerol stocks prepared.

Fermentation Medium.

UT5600_M9_YE medium was prepared by mixing, for the batch media: 16grams/L Yeast Extract (Difco 212750), 8 g/L Glycerol (Sigma G2025),11.28 g/L M9 Salts (Difco 248510) and 100 μl/L Antifoam 204 (SigmaAG6426) to Deionized water and sterilized via autoclave. Post autoclaveadditions were 0.64 ml/L Trace Metals Solution, 2.3 ml/L 100× MagnesiumSulfate and 45.83 g/L L-Leucine. Feed Media was prepared by mixing 250g/L Yeast Extract, 225 g/L Glycerol and 100 μl/L Antifoam 204 todeionized water and sterilized via autoclave. Post-sterilizationadditions were 10 ml/L Trace Metals Solution, 2.3 ml/L 100× MagnesiumSulfate, 45.83 ml/L L-Leucine.

Fed-Batch Fermentation.

A 0.5 L fermentor (Infors) with MFCS/DA software was used for thefed-batch fermentation. The agitation was set to cascade at 500-1200rpm. The pH value was controlled at 7.0±0.1 automatically by theaddition of 30% ammonium hydroxide (Sigma 221228) and 30% phosphoricacid (Sigma P5811). The air was provided at a flow rate of 0.5 L/minwith an oil-free diaphragm air compressor (Cole-Parmer). The air waspassed through a 0.2 μm Midisart 2000 filter (Sartorius 17805). The pureoxygen (West Air) was supplied automatically to control the dissolvedoxygen level at 30%. The temperature was controlled at 30° C. with aNeslab RTE7 circulator (Thermo Scientific). The foaming was controlledby addition of the antifoam 204 (Sigma A8311) as needed. The initialvolume of UT5600_M9_YE medium in the fermentor was 0.24 L. The fermentorwas inoculated with ≈10 OD Units (approximately 1-2 ml of seed at OD5-10) of the seed culture grown for 6 hours at 37° C. and 250 rpm. Whenthe batch glycerol was depleted in the vessel (˜4 hours), theconcentrated feeding solution was introduced into the vessel by aperistaltic pump set on an exponential feeding program. When the opticaldensity of the cells at 600 nm reached ˜150, the culture was inducedwith 0.5 mM IPTG (Fisher Scientific BP1755). The culture was incubated 4hours post induction and harvested by centrifugation at 10,000×g for 30minutes. Approximate final cell pellet yield was 150-200 grams per literwet cell weight (WCW). The cell pellet was stored at −80° C. untilpurification. The expression of target protein was confirmed viaSDS-PAGE and western blot to Goat anti-Human IgG, HRP conjugatedantibody (Thermo p/n 31413).

Purification of FC Fusion Proteins.

Frozen cell pellets were resuspended in 4 volumes (i.e., 4 mL/g cellpellet) of Lysis Buffer (50 mM Tris, 500 mM NaCl, 14 mM β-ME, pH 7.5).Complete EDTA-FREE protease inhibitor tablets (Roche) were added to thesuspension at a ratio of 1 tablet/50 mL. The suspension was passedthrough a microfluidizer (Microfluidics) twice at 14,000 psi withcooling by ice. The lysate was centrifuged at >10,000×g for 45 min at 4°C. The supernatant was filtered through 0.45+0.22 μm Sartobran capsulefilters (Sartorius).

The clarified lysate was bound to the MabSelect resin (GE Healthcare),pre-equilibrated with Binding Buffer (50 mM Tris, 500 mM NaCl, pH 7.5)at a ratio of 1 ml resin per 10 g cell paste. The column was washed with500 column volumes of Binding Buffer+0.1% Triton X-114 followed by 100column volumes of the Binding Buffer. The bound protein, fusion proteinswere eluted with 3.75 column volumes of Elution Buffer (0.1 M glycine,0.5 M Arginine, pH 3.0) to a collection tube containing 1.25 columnvolumes of Neutralization Buffer (1 M Tris, pH 8.0).

Optionally, for further removal of high molecular weight species thematerial was concentrated in Amicon 30 kDa ultracentrifugalconcentrating devices (Millipore) and loaded onto a HiLoad Superdex 200pg 16/600 size-exclusion chromatography column (GE Healthcare). Thematerial was eluted in 1.1 column volumes of 1×PBS pH 7.4 (Gibco#10010), and fractions corresponding to the main peak based on theprocess chromatogram absorbance at 280 nm were pooled.

If size-exclusion chromatography was not performed, the purified Fcfusion proteins were buffer exchanged into a buffer containing PBS, atpH 7.4. The dialyzed protein was passed through a Q membrane filter(Sartobind-Q from Sartorius or Mustang-Q from Pall) or a Q-Sepharosecolumn (GE Healthcare) for further endotoxin removal, and filteredthrough a 0.22 μm sterile filter.

Scaleable Purification Process for FC Fusion Proteins.

The purification process using MabSelect followed by size-exclusionchromatography was used to purify multiple Fc fusion proteins using arobust process effective with minimal purification development. However,the use of detergent wash during Protein A (MabSelect) and asize-exclusion chromatography step limits the ability to scale uppurification. A purification process for scale-up was also developed forFc fusion proteins using lysate flocculation, Protein A chromatography,cation exchange (CEX), and ceramic hydroxyapatite (CHT) chromatography.

Resuspension, and lysis were performed as described above, with theomission of protease inhibitor tables and β-ME from the Lysis Buffer.After lysis, the lysate was flocculated with addition ofpolyethyleneimine, Mw 1300 (Sigma Aldrich) to 0.04% (v/v) and incubatedfor 30 min @ 4° C. Centrifugation and clarification were performed asdescribed above. Protein A chromatography was performed on clarifiedlysate using MabSelect resin in a packed chromatography column at a loadratio of 1 ml resin per 4 g cell paste, with a wash step of 5 columnvolumes in 50 mM Tris, 500 mM NaCl pH 7.5, followed by elution in 3column volumes of 0.1 M glycine pH 3.0 and neutralization in 0.3 columnvolumes of 1 M Tris pH 8.0. Following Protein A, CEX load was preparedby 5× dilution of post-Protein A eluent in CEX Equilibration buffer (20mM sodium phosphate, pH 6.0), loaded onto a SP Sepharose HighPerformance column, washed with 5 column volumes of Equilibrationbuffer, and eluted over a linear sodium chloride gradient from 0 to 300mM NaCl over 10 column volumes. CEX fractions were pooled based onSDS-PAGE analysis of elution peak fractions. Ceramic hydroxyapatite wasperformed on CEX pool by loading onto a CHT Type I 40 μm column(Bio-Rad) equilibrated in CHT Equilibration Buffer (5 mM sodiumphosphate, 150 mM sodium chloride, 1 μM calcium chloride pH 6.5), washedwith 5 columns of equilibration buffer and eluted over a linear sodiumchloride gradient from 150 mM to 1.5 M sodium chloride over 10 columnvolumes and a 1.5 M NaCl hold for up to 20 column volumes to completethe elution. Following CHT, protein was buffer exchanged into 1×PBS pH7.4 using Amicon 30 kDa centrifugal concentrating devices (Millipore).

The fusion protein concentration was determined by Bradford proteinassay (Thermo Scientific) or by UV absorbance at 280 nm. The fusionprotein concentration was determined by Bradford protein assay (ThermoScientific). The endotoxin level was below 4 EU/mg as determined byEndoSafe PTS LAL assay (Charles River).

Analysis of HRS-Fc Fusion Proteins.

The purified HRS-Fc fusion proteins were analyzed by SDS-PAGE as shownin FIG. 21. Samples of 10 μg protein load were run on NuPAGE 4-12%Bis-Tris gels, 150 V for 60 minutes in MOPS-SDS buffer, and stained withInstant Blue. Reduced samples had 25 mM DTT, and were heated at 95° C.for 10 minutes in 1×LDS buffer prior to loading.

The purified HRS-Fc fusion proteins were also analyzed by asize-exclusion chromatography (SEC) method. The samples were loaded toaTSK-Gel Super SW3000 column (TOSOH, 4.6 mm ID×30 cm, 4 μm) on anAgilent 1260 HPLC system. A 30 minute isocratic run was carried out at0.3 ml/min with a mobile phase containing 0.1 M NaCl, 0.2M Na phosphateand 5% 2-propanol at pH7. UV detection was performed at 280 nm. Thechromatogram is shown in FIG. 22.

Approximately 83% of the protein is in the desired dimer form. afterProtein A and prior to size-exclusion chromatography purification, withthe remaining quantity present as high molecular weight species. Aftersize-exclusion chromatography, the proportion of dimer increases to 95to 99%. Using the Protein A, cation exchange, and hydroxyapatitepurification process the proportion of dimer is greater than 99%. Mostof the dimer protein contains the inter-chain disulfide bond in the Fchinge region, while some non-covalent dimer also exists.

Analysis of the intact mass spectral data obtained using LC/ESI-MSdemonstrates that the molecular size of the FC fusion proteins undernon-reducing conditions is consistent with the expected molecular massof approximately 64,520 daltons (data not shown). The CD spectra of theFc fusion proteins in the far and near UV regions reveals that thestructure of the fusion proteins is consistent with the expected domainstructures. Additionally the deconvoluted differential scanningcalorimetry data obtained from the HRS-Fc fusion proteins demonstratesthat the Fc fusion proteins are folded with two major thermaltransitions characteristic of the CH1 and CH2 domains of the Fccomponent (data not shown), consistent with predicted structures.

To assess the pharmacokinetic characteristics of the HRS-Fc fusionprotein constructs compared to the unmodified HRS proteins, proteinswere administered to normal C57BL/6 mice via a single intravenous orsubcutaneous bolus at a dose of 8 mg/kg. Blood was serially sampled withsampling times distributed across nine animals per product form. Foreach time point, sera from three independent mice were drawn. Testarticle concentrations were measured by ELISA and pharmacokineticparameters were derived using non-compartmental analysis on Phoenixsoftware.

The results, shown in FIGS. 23A, 23B, and 23C demonstrate that thecreation of the Fc fusion proteins resulted in significantly enhancedhalf life, exposure and SC bioavailability

TABLE E16 Pharmacokinetic analysis of HRS-Fc fusion proteins Fold V_(d)¹ CL² Half-life Bioavailability³ AUC⁴ increase in Product Form Route(ml/kg) (ml/hr/kg) (hr) (%) (hr*nM) exposure ⁵ HRS(1-60) IV 119 574 0.5— 1,835 — Fc-HRS(2-60) IV 176 2.8 72 — 89,916 209 HRS(1-60)-Fc IV 1275.1 33 — 49,364 115 HRS(1-60) SC 1,457 1,541 0.7 37 683 Fc-HRS(2-60) SC452 4.4 71 63 56,307 352 HRS(1-60)-Fc SC 537 10.2 37 50 24,623 154¹Volume of distribution at steady state for IV administration orterminal phase for SC administration ²Clearance for IV administration orclearance as a function of bioavailability for SC administration³Compared to same product form administered IV ⁴Area under the curvefrom time of dose predicted to infinity ⁵ Fold increase in molarexposure compared to unmodified protein delivered the same route

The pharmacokinetic analysis shown in Table E16 demonstrates that HRS-Fcfusion constructs exhibit significantly improved systemic exposure,clearance and half-life compared to the unmodified proteins. Creation ofthe Fc fusion proteins also improved the subcutaneous bioavailability ofthe proteins compared to the unmodified proteins. In particularFc-HRS(1-60) increased exposure compared to the unmodified protein by200 to 300 fold depending on the route of administration, additionallySC bioavailability and half life were both significantly enhanced.

Example 9 Testing of Fc Fusion Proteins in TNBS Induced Colitis

The large intestine is lined by an epithelial mucosa that is invaginatedinto flask-like structures, crypts. Unlike the small intestine there areno villi in this region, with the top of the crypts opening onto a flattable region. The cells of the crypt are generated by stem cells locatedat the crypt base, whose daughter cells divide rapidly and differentiateinto the predominant colonocytes and mucin producing goblet cells (asmaller number of endocrine cells and M cells are also produced). Thesize of the crypt and the number of goblet cells per crypt increasealong the large intestine from caecum to rectum, presumably to aid thepassage of faeces and provide sufficient mucosal and stem cellprotection as water is absorbed from the faeces.

Normally, the rate of cell production in the intestinal crypt isprecisely matched to the rate of cell loss—a very sensitive homeostaticmechanism operates. Disruption of the mucosal barrier allows bacterialentry into the body, with resultant disease implications. Conversely,hyperplasia can generate polyps and ultimately tumours. Disruption ofthe intestinal barrier can be caused by exposure to non-cell typespecific (often proliferation specific) cytotoxic agents—typicallyanti-cancer therapies. However, perturbation of epithelial cell turnoveris also a common feature of inflammatory diseases.

Current rodent models of inflammatory bowel disease (IBD) include forexample models generated by triggering an autoimmune disease bymanipulation of the T-cell population, irritating the mucosal lining ofthe intestine by the accumulation of particulate material in the largebowel (such as with DSS, dextran sulphate sodium), or chemicaldisruption of the epithelium (such as with Trinitrobenzene sulfonate,TNBS).

In any of these models disease severity may be assessed by a variety ofsubjective assessments of the observed pathological grades, as well asmore objective and quantitative measures of the damage, to provide moremeaningful insights into the underlying biology. It is possible tobroaden the analysis by using computer assisted length/area measurementsto map the changes in the mucosa/submucosa and obtain more quantitativemeasures of the damage, and hence therapeutic efficacy. While each ofthese models as different pros and cons, the TNBS colitis model is anestablished model of various aspects of inflammatory bowel disease inhumans, which has been successfully used to validate and optimize theefficacy of human therapeutics.

TNBS Mouse Model.

In this model of colitis, colonic irritation is induced by intracolonicadministration of TNBS in ethanol. This provokes an acute colitis thathas a TH1-type cytokine profile, which is characterised by theexpression of genes coding for TNF-α, IFN-γ and IL-12 amongst others(Fichtner-Feigl et al. J. Clin. Invest. 2005. 115: 3057-3071). Thecolitis can be severe and localised to the area of the colon into whichthe TNBS is introduced. The inflammatory response results in localisedswelling, inflammatory cell infiltration, and epithelial loss.

In this study, the efficacy of the unmodified HRS polypeptide,(HRS(1-60), was compared to the Fc fusion proteins Fc-HRS(2-60) andHRS(1-60)-Fc to assess their efficacy in ameliorating TNBS-induced acutecolitis in mice. Three different dosing regimens, employing either i.v.or s.c. administration of test item, were evaluated. Budesonide (p.o.)was used as a reference item for the study.

Animals and Caging:

A total of 100 BDF-1 (H. Pylori-free, murine norovirus-free) male mice(Harlan Laboratories, UK) were used in the study. Animals were 8-10weeks old on supply and used at 10-12 weeks of age. All mice were heldin individually ventilated cages (IVCs) in an SPF (Specific PathogenFree) barrier unit. The animals were identified by numbered cages and byear punches.

Diet and Animal Welfare: The animals were fed Rat and Mouse Expandeddiet from B & K.

Water was supplied in HYDROPAC™ water pouches (filtered RO water;Hydropac/lab products, Delaware, USA). Both feed and water wereavailable ad libitum. There was a constant room temperature of 21±2° C.and a mean relative humidity of 55±10%. The day-night cycle wasconstant, with light and dark phases of 12 hours each (07:00 hr/19:00 hrswitch). Animal health was monitored daily and cages were cleaned atregular intervals. All procedures were certified according to the UKAnimal (Scientific Procedures) Act 1986.

Groups, Dosages, Administration and Formulations:

A total of 100 mice were randomised into ten study groups (Table E17).All the mice in any one cage received the same treatment and were earpunched for identification purposes. Daily body weight measurements wereused to calculate the volume of test item or vehicle administered to theapplicable groups.

TABLE E17 Study groups Volume and Fre- Dura- Dose of route of quencytion test adminis- of of Mouse Treatment item tration dosing dosingcodes Vehicle (PBS) — 5 ml/kg, i.v. q.d. day 0 to 1-6 only day 3TNBS/vehicle — 5 ml/kg, i.v. q.d. day 0 to  7-18 (PBS) day 3 TNBS + — 5ml/kg, i.v. q.d. day 0 to 19-28 budesonide day 3 TNBS + 1 mg/kg 5 ml/kg,i.v. q.d. day 0 to 29-40 HRS(1-60) day 3 TNBS + Fc- 5 mg/kg 5 ml/kg,i.v. q.d. day 0 to 41-50 HRS(2-60) day 3 TNBS + Fc- 5 mg/kg 5 ml/kg,i.v. once only day 0 51-60 HRS(2-60) TNBS + Fc- 15 mg/kg  5 ml/kg, s.c.q.d. day 0 to 61-70 HRS(2-60) day 3 TNBS + 5 mg/kg 5 ml/kg, i.v. q.d.day 0 to 71-80 HRS(1-60)-Fc day 3 TNBS + 5 mg/kg 5 ml/kg, i.v. once onlyday 0 81-90 HRS(1-60)-Fc TNBS + 15 mg/kg  5 ml/kg, s.c q.d. day 0 to 91-100 HRS(1-60)-Fc day 3

Preparation and Administration of TNBS and Test Items.

TNBS: TNBS (Sigma; lot #SLBD6811V) was prepared as a 15 mg/ml solutionin saline/50% ethanol. A single dose of 200 μl (3 mg TNBS) was instilledinto the colon, using a plastic catheter, placed 4 cm proximal to theanal verge, at 11:00 hr on study day 0. Animals were maintained in aninverted position for 1 minute after introduction of TNBS into thecolon, in order to minimise leakage of the compound.

HRS(1-60):

The test item was received as four vials of frozen stock solution (0.033ml volume in each) at 17.1 mg/ml, which was stored at −80° C. until use.On each day of test item administration, a single aliquot was taken andthawed on wet ice. After thawing, the contents of the vial were mixed bypipetting them up and down ten times. Subsequently, the test item wasdiluted with cold vehicle (sterile, ×1 PBS, pH 7.4) to give a solutionat 0.2 mg/ml; the solution was mixed by pipetting up and down ten times.This solution was administered daily (from study day 0 until study day3) by intravenous injection, at 5 ml/kg, in order to give a dose of 1mg/kg.

Fc-HRS(2-60):

The test item was received as four vials of frozen stock solution (onevial of 2.51 ml and three vials of 2.01 ml) at 4.7 mg/ml, which wasstored at −80° C. until use. On each day of test item administration, asingle aliquot was taken and thawed on wet ice. After thawing, thecontents of the vial were mixed by pipetting them up and down ten times.Subsequently, the test item was diluted with cold vehicle (sterile, ×1PBS, pH 7.4) to gives solutions at 3 mg/ml and 1 mg/ml; the solutionswere mixed by pipetting up and down ten times. Fc-HRS(2-60) wasadministered according to 3 different regimens: i) the 1 mg/ml solutionwas administered daily (from study day 0 until study day 3) byintravenous injection, at 5 ml/kg, in order to give a dose of 5 mg/kg;ii) the 1 mg/ml solution was administered daily, once only on study day0, by intravenous injection, at 5 ml/kg, in order to give a dose of 5mg/kg; iii) the 3 mg/ml solution was administered daily (from study day0 until study day 3) by subcutaneous injection, at 5 ml/kg, in order togive a dose of 15 mg/kg.

HRS(1-60)-Fc:

The test item was received as four vials of frozen stock solution (onevial of 2.37 ml and three vials of 1.89 ml) at 4.99 mg/ml, which wasstored at −80° C. until use. On each day of test item administration, asingle aliquot was taken and thawed on wet ice. After thawing, thecontents of the vial were mixed by pipetting them up and down ten times.Subsequently, the test item was diluted with cold vehicle (sterile, ×1PBS, pH 7.4) to gives solutions at 3 mg/ml and 1 mg/ml; the solutionswere mixed by pipetting up and down ten times. HRS(1-60)-Fc wasadministered according to 3 different regimens: i) the 1 mg/ml solutionwas administered daily (from study day 0 until study day 3) byintravenous injection, at 5 ml/kg, in order to give a dose of 5 mg/kg;ii) the 1 mg/ml solution was administered daily, once only on study day0, by intravenous injection, at 5 ml/kg, in order to give a dose of 5mg/kg; iii) the 3 mg/ml solution was administered daily (from study day0 until study day 3) by subcutaneous injection, at 5 ml/kg, in order togive a dose of 15 mg/kg.

Budesonide:

Budesonide was obtained from Tocris (Tocris 1101, lot #1A/128902) andwas stored in the dark at ambient temperature until use. On each day ofadministration, budesonide was formulated as a 1 mg/ml solution inpeanut oil (Sigma). Budesonide was administered daily (from study day 0until study day 3) by oral gavage, at 5 ml/kg, in order to give a doseof 5 mg/kg.

Clinical Examinations and Analgesia.

Any animal demonstrating more than 15% weight loss was considered unwelland treatment may have been withheld. Any animal was culled if theweight loss was greater than 20%. Animal well-being was monitored daily.Once daily from day −1 until the end of the study, all mice were weighedand assessed for stool consistency, and the presence of overt blood inthe stool or around the anus according to the criteria in Table E 18.

TABLE E18 Scoring criteria for in-life disease parameters. Overt bloodWeight Loss (in stool/ Score (% day 0 weight) Stool observation aroundanus) 0 <1% Normal None 1 ≥1% <5% Soft; empty colon/rectum Slight atnecropsy; No observation in a 30 minute period 2  ≥5% <10% UnformedModerate 3 ≥10% <15% Watery/gel-like Severe 4 ≥15% 

Use of Analgesia:

Analgesia was not used in this study, at the direction of the Sponsor,as it may have interfered with test item action.

Harvesting and Preparation of Tissue for Histological Examination:

Upon sacrifice, mice were anaesthetised with 4% isofluorane, with 2L/min 02 and 2 L/min N20. When fully anaesthetised, blood was withdrawnby direct cardiac puncture and death confirmed by cervical dislocation.

Preparation of Whole Blood and Plasma Samples.

Blood was collected by cardiac puncture from all mice into 1.5 mlmicrocentrifuge tubes. Blood samples were immediately placed on ice andleft to clot for 60 minutes. Samples were then centrifuged at 3000 g for7 minutes, at 4° C. Immediately after centrifugation, the serum wastransferred by sterile pipette into pre-labelled vials and immediatelyfrozen on dry ice.

Preparation of Intestinal Samples.

The large intestine was removed and flushed with PBS and its length andwet weight were recorded, prior to cutting into proximal, mid and distalregions and fixation in Carnoy's solution. In addition, a small sampleof colon was snap-frozen in liquid nitrogen. Fixed tissue was dehydratedthrough a series of alcohols and xylene and embedded in paraffin, usinga Leica TP1020 tissue processor and an EG1140H work station. Sections (5μm thick) were cut using a Leica RM2125RTF microtome, and air-dried onto microscope slides, overnight at 37° C. Subsequently, slides weredewaxed in xylene and rehydrated through graded alcohols to PBS. Allsections were then stained with haematoxylin and eosin (H&E), andmounted.

Histological Analysis:

Histological sections were assessed in a blinded fashion. Sections wereobserved microscopically, and assigned a subjective severity scoreranging between 0 and 5, according to the criteria outlined in TableE19. Up to twelve transverse cross-sections from the mid and distallarge bowel were assessed.

TABLE E19 Epistem's standard severity scoring system. Severity ScoreDescription 0 Crypts appeared normal. 1 Crypts present but damaged(abnormal pathology). No ulceration. 2 Some crypts depleted and someulceration/inflammation. 20-70% of crypts depleted and increasedulceration/inflammation. 4 >70% crypts depleted with substantialulceration/inflammation. 5 No crypts remaining. Totallyulcerated/inflamed.

Statistical Analysis:

Where mentioned, statistical comparisons of group data were performedusing ANOVA, in combination with post hoc tests, using Graph Pad Prism.

qPCR Analysis:

Mouse colon was excised from the animals and stored at −80° C. untilanalysis. RNA was prepped from colons using Qiagen's RNeasy Mini Kit(Catalog #74106). Once RNA was eluted from the Qiagen column, it was runon an Agilent Bioanalyzer 2100 to test RNA integrity and NanoDrop todetermine RNA concentration and purity. RNA was then stored at −80° C.

Reverse transcription (RT) of RNA to cDNA was performed in a 96 well PCRplate format in Eppendorf's Mastercycler PCR machine with the followingprogram: 37° C. for 60 minutes, 95° C. for 5 minutes. The edge wells ofthe 96 well plate were not used and filled with 50 mcL water to preventevaporation of inside wells. 100 ng of RNA in 25 mcL of reversetranscription master mix (Life Technologies #4387406) was used persample RT. Once RT was completed, the next step was to pre-amplify genesof interest in the sample cDNA. Primers of genes of interest (DELTAgeneprimers designed by Fluidigm) were combined to a final concentration of200 nM. Using these primers, genes of interest were pre-amplified ineach sample. Pre-amplification was performed in 10 mcL reactions (2.5mcL cDNA, 7.5 mcL Pre-Amp mastermix) in 384-well format using an AppliedBiosystems ViiA7 PCR machine with the following program: 95° C. for 10minutes, 14 cycles of 95° C. for 15 seconds and 60° C. for 4 minutes.After pre-amplification step, exonuclease (New England BioLabs catalog#M0293L) was added to remove unincorporated primers from each sample.This exonuclease reaction was also completed in the ViiA7 PCR machinewith the following program: 37° C. for 30 minutes, 80° C. for 15minutes. After exonuclease, the RT sample was further diluted 1:5 (7 mcLexonuclease sample+18 mcL low EDTA buffer).

The chip used to run qPCR on Fluidigm's Biomark system was a 96.96Dynamic Array IFC for Gene Expression. The chip was first primed withthe IFC controller HX as per manufacturer's recommendations beforesample and assays were loaded. To prepare assays to be loaded on a chip,4.4 mcL assay master mix (Fluidigm's 2× Assay Loading Reagent catalog#8500736 and low EDTA TE) to 3.6 mcL 20 mcM forward and reverse primersfor each gene of interest were prepared in a 96 well plate. To preparesamples, 4.5 mcL sample master mix (Ambion's 2× TaqMan Gene ExpressionMaster Mix, Fluidigm's 20×DNA Binding Dye Sample Loading Reagent catalognumber 100-0388, and Biotium's 20× EvaGreen catalog #31000) was added to3 mcL diluted pre-amplified/exonuclease sample in a 96 well plate. Oncethe chip had been primed, 5 mcL sample or assay prepared above wereloaded onto the chip. The chip was them returned to the IFC controllerfor the samples to be loaded into the chip. After the chip had finishedloading, qPCR could then be run on the Biomark using preset program for96.96 Dynamic Array for Gene Expression with a melt curve to determineprimer specificity. Relative gene expression was determined by the deltadelta Ct method using multiple housekeeping genes.

Results:

In-Life Parameters—

All TNBS-recipient groups demonstrated an acute decrease in mean bodyweight, of between 8-10% of starting body weight over the first 24 hoursfollowing administration of the colitic agent; subsequently, there was aslower decrease in mean body weight, with all groups showing asignificant decline in mean body weight to between 80-90% of startingweight by the end of the study (p<0.0158). The body weight ofvehicle-only treated mice showed a minimal change over the period of thestudy, with a final mean body weight of 99.6±1.7% of starting bodyweight, by study day 4 (mean±standard deviation). The TNBS/vehicle grouphad the lowest mean final body weight, at 79.8±1.8%. Final mean bodyweights for some groups (i.e. TNBS/Fc-HRS(2-60)-s.c. group andTNBS/budesonide group) were artificially high, due to the higherincidence of early morbidity in these groups. Of the groups thatdemonstrated the least early morbidity, the TNBS/Fc-HRS(2-60)-QD -i.v.group had the highest mean final body weight of 85.6±7.1%, although thiswas not significantly different from the TNBS/vehicle group (p>0.05)(data not shown).

The majority of TNBS-recipient mice demonstrated diarrhoea at some stageduring the study, although observations of mucosal bleeding were lesscommon. There were reductions in the incidence of diarrhoea in responseto some test item treatments. The TNBS/vehicle group had a cumulative(normalised) diarrhoea score of 31 by the end of the study on day 4;with the TNBS/Fc-HRS(2-60)-QD-i.v. and TNBS/HRS(1-60)-Fc-QD-i.v. groupshaving the lowest scores of 26 (data not shown).

The clinical condition of the mice can be best obtained by combininginformation on bleeding and diarrhoea together with a score for weightloss, in order to give a disease activity index (DAI) score. This can bemost accurately determined on the day that a mouse is euthanised, asmissing stool consistency observation data can be supplemented withobservations of stool in the rectum. For mice surviving on day 4, therewas a significant increase in the mean DAI scores for all theTNBS-recipient groups, in comparison to the untreated control group (atp<0.0062), except for the TNBS/Fc-HRS(2-60)-s.c. group, although in thisgroup only three mice remained in the study at the scheduled end point.The mean DAI for surviving mice on day 4 in the TNBS/vehicle group was7.13±0.64; in comparison, the TNBS/budesonide andTNBS/HRS(1-60)-Fc-once-i.v. groups had significantly lower mean DAIs, at4.33±1.97 and 4.20±3.03, respectively (p<0.0304). Inclusion of all mice,surviving to at least 07:00 hr on day 1, in the calculation of DAI gavesimilar results, although the effect observed in the TNBS/budesonide andTNBS/HRS(1-60)-Fc -once-i.v. groups was no longer significant (p>0.05)(see FIG. 24A).

Post Mortem Observations—

For the vehicle-only mice, mean large bowel weight and length were200.4±21.7 mg and 108.3±7.8 mm, respectively; the mean large bowelweight:length ratio was 1.85±0.12 (mg/mm). All TNBS-treated groupsdemonstrated a significant increase in large bowel weight relative tothe untreated control mice (p<0.0215), with the TNBS/vehicle grouphaving a mean large bowel weight of 368.4±70.1 mg. The TNBS/budesonidegroup had the lowest mean large bowel weight of all the TNBS-recipientgroups, at 298.0±62.0 mg. TNBS administration was associated also with asignificant reduction in large bowel length in all TNBS-recipient groups(p<0.0285), with the TNBS/vehicle group having a mean large bowel lengthof 75.6±9.3 mm; in comparison, the TNBS/HRS(1-60)-Fc-QD-i.v. groupdemonstrated a significant amelioration of large bowel shortening, witha mean large bowel length of 94.9±11.8 mm (p=0.0019). The changes inlarge bowel weight and length resulted in a significant increase inlarge bowel weight:length ratio for all TNBS-recipient groups(p<0.0116). The TNBS/vehicle group had the highest mean large bowelweight:length ratio of all groups, at 4.92±0.99 (mg/mm). TheTNBS/budesonide group, TNBS/Fc-HRS(2-60)-s.c. group, TNBS/HRS(1-60)-Fc-QD-i.v. group and the TNBS/HRS(1-60)-Fc -once-i.v. group alldemonstrated a significant reduction in large bowel weight:length ratio,relative to the TNBS/vehicle control group, although the mean ratio forthe TNBS/HRS(1-60)-Fc -QD-i.v. group was group was skewed by the numberof mice that were euthanised early in the study. The mean large bowelweight:length ratios for the TNBS/budesonide and TNBS/HRS(1-60)-Fc-QD-i.v. groups were 3.57±1.01 and 3.56±0.80, respectively (p=0.0143 andp=0.0456) (see FIG. 24B).

Histopathology—

Histopathological changes associated with TNBS-induced colitis wereassessed according to Epistem's standard histological scoring procedure.All TNBS-recipient groups demonstrated a significant increase inhistopathology score (p<0.0036). The T cell/vehicle group had a meanhistopathology score of 2.32±0.65, with the TNBS/HRS(1-60)-Fc -s.c.group having the highest mean histopathology score at 2.41±0.68. Othertreatment groups had lower mean histopathology scores than theTNBS/vehicle group, with the TNBS/budesonide andTNBS/Fc-HRS(2-60)-QD-i.v. groups having the lowest scores of 1.66±1.08and 1.68±1.03, respectively, although these reductions were notstatistically significant. On a regional basis, the effect of thesetreatments was more apparent in the distal third of the large bowel,although still not of statistical significance.

Morbidity—

Only one group, TNBS/HRS(1-60)-Fc -QD-i.v. demonstrated survival of all(eligible) mice (mouse 75 excluded due to large bowel perforation duringTNBS administration and mouse 78 excluded due to lack of diseaseinduction). Survival in other groups ranged from 80%(TNBS/Fc-HRS(2-60)-QD-i.v. group) to 33.3% (TNBS/Fc-HRS(2-60)-s.c.group); survival in the TNBS/budesonide group was 60%. Mice wereeuthanised when they presented with poor condition (withdrawn/failure todemonstrate normal behaviour); despite six-hourly checks, two mice (40and 50) were found dead at 19:00 hr on study day 3 (data not shown).

qPCR Results.

To further investigate the mechanistic basis for the effects ofHRS(1-60) and Fc-HRS(2-60) on TNBS induced colitis, changes in geneexpression in the colons from animals was examined after the completionof the study. RNA profiling was performed on colons isolated from themice on day as described above. The results from these studiesdemonstrated that seven genes were elevated by more than 10 fold inresponse to TNBS treatment were significantly reduced by treatment withFc-HRS(2-60) (see Table E20; and FIG. 25).

TABLE E20 Genes regulated by more than 10 fold IL6 IL1b MCP1 MMP3 MMP9CD11B IL10

Transcriptional profiling of TNBS treated mouse colons revealed manygenes, including several housekeeping genes (data not shown) were notsignificantly impacted by Fc-HRS(2-60) treatment. By contrast,transcriptional profiling of TNBS treated mouse colons revealedsignificant reduction of TNBS regulated immune and inflammatory relatedgenes by Fc-HRS(2-60). This result was fortified by the finding thatHRS(1-60) also significantly reduces TNBS induced levels of MCP1, MMP3,CD11b, and IL10 (see FIGS. 26A-26H).

Conclusion:

Intracolonic administration of TNBS to BDF-1 mice resulted in thedevelopment of colitis characterised by acute weight loss, with amoderate increase in the incidence of diarrhoea and mucosal bleeding.Post mortem examination demonstrated significant change in the largebowel weight:length ratio and ulcerative lesions in the large bowel,with stenosis and accompanying stool accumulation within the bowel.Treatment with budesonide was associated with improvements in bothin-life and post mortem disease parameters, and although having thelowest mean histopathology score of all TNBS-recipient groups, thereduction in this disease parameter did not achieve significance.Treatment with Fc-HRS(2-60)-QD-i.v. had a similar effect to budesonidein reducing histopathology score and was associated with superiorsurvival. The TNBS/HRS(1-60)-Fc -QD-i.v. group showed the highest levelof survival and significant improvement in large bowel weight:lengthratio, although it was less effective than either budesonide orFc-HRS(2-60) in reducing the histopathology score. With regard tohistopathology score, all three of these test items appeared to havetheir greatest effect on the distal third of the large bowel.Improvements in disease parameters were observed for theTNBS/Fc-HRS(2-60)-s.c. group, and the TNBS/HRS(1-60)-Fc -once-i.v. group(e.g. body weight and large bowel weight:length ratio). Additionally,transcriptional profiling of TNBS treated mouse colons revealedsignificant reduction of TNBS regulated immune and inflammatory relatedgenes by Fc-HRS(2-60). This result was fortified by the finding thatHRS(1-60) also significantly reduces TNBS induced levels of MCP1, MMP3,CD11b, and IL10, demonstrating the the HRS-Fc fusion proteins wereactive in immunomodulating gene expression in this system.

Overall, the data suggest that Fc-HRS(2-60) and HRS(1-60)-Fc havesignificant potential in the treatment of intestinal inflammation, andother inflammatory conditions.

Example 10 Impact of Hrs-Fc Fusion Proteins on T Cell Populations

To evaluate the potential impact of HRS-Fc conjugates on T cellpopulations in vivo, Fc-HRS(2-60) was tested in a TNBS induced model ofcolitis, similar to that described above. Studies were performed in maleBDF-1 mice. Briefly, male BDF-1 mice (Jackson Laboratories), wereacclimated for a minimum of 1 week prior to experimentation, were fastedbeginning 16 hours prior to TNBS administration and 13 hours prior todosing. Fc-HRS(2-60) was administered at a single dose, 0.5 mg/Kg, IV atthe same time as commencing TNBS treatment. Splenocytes from threeanimals per group were analyzed for immune populations. To choose theanimals for analysis, the mice that had the most severe and the leastsevere disease scores were excluded (based on clinical observations,stool consistency and body weight). From the remaining animals, threeanimals from each treatment group were picked based on representing themean of their respective group.

Procedures.

Spleens were harvested from BDF-1 mice and placed directly into 10 mlcold Cell Staining Buffer (Biolegend, catalog #420201, lot #B166478) onice. Spleens were mechanically disassociated between two frostedautoclaved slides in a tissue culture dish. The cell suspension wasfiltered through a 70 μm filter and cells were pelleted bycentrifugation at 300 g for 5 minutes, 4° C. Cells were washed once in10 ml Cell Staining Buffer and counted using the Nexcelom CellometerAuto 2000 Cell Viability Counter. Splenocytes were reconstituted at5×10⁵ cells/ml in Cell Staining Buffer and were immediately stained withantibodies for flow cytometry.

A total of 1×10⁶ cells were stained for T regulatory markers (CD4, CD25and FOXP3) using the One Step Staining Mouse Treg Flow Kit (Biolegendcatalog #13680, lot numbers #B177852, B177853, B174309, B176365)according to manufacturer's instructions.

For phenotyping of immune cells, a total of 1×10⁶ cells were stainedwith fluorescently-labeled antibodies against CD3ε (catalog #100334, lot#162956), CD4 (catalog #100529, lot #B152907) and CD8 (catalog #100714,lot #B165226) (antibodies were purchased from Biolegend). Cells werestained with antibodies diluted at manufacturer's recommendedconcentrations in Cell Staining Buffer and incubated for 30 minutes at4° C. Cells were thoroughly washed with Cell Staining Buffer and fixedusing Stabilizing Fixative Buffer (BD Biosciences catalog #338036, lot#2291614).

Flow cytometry was performed at the VA Flow Cytometry Research Corefacility (VA San Diego, La Jolla, Calif.). The samples were analyzed ona 3 laser BD Canto instrument (488 Argon, 633 HeNe, 405 Violet). RawFACS files were analyzed using FlowJo software (Tree Star).

Results.

Extracellularly-stained splenocytes were gated on a live lymphocytepopulation and CD3⁺ cells to determine a total T cell percentage. Togenerate population percentages of CD4⁺ and CD8⁺, cells were gated intoCD3⁺CD4⁺ T cells and CD3⁺CD8⁺ T double-positive cells from the livelymphocyte gate. T regulatory cells (Treg) were gated on a livelymphocyte gate and then on CD4⁺ cells. Treg cells were determined basedon expression of CD25 and FoxP3. In these data (see FIGS. 27A-27D), eachdot represents one animal, the line represents the mean.

In TNBS-treated mice, there was an increase in CD3⁺ T cells (FIG. 27A),compared to naïve animals. By contrast, two out of the threeFc-HRS(2-60) treated mice showed a reduction in CD3⁺ T cells compared tothe TNBS treated animals. To determine which CD3⁺ populations wereaccounting for this change, CD8⁺, CD4⁺ and Treg cells populations werefurther investigated. FIG. 27B and FIG. 27C show that CD8⁺ T and CD4⁺cells were elevated in TNBS-treated animals and treatment withFc-HRS(2-60) reduced levels in both cases. Furthermore, Treg cells weredepleted in TNBS-treated animals, compared to naïve animals, but wereelevated compared to the TNBS treated animals in the Fc-HRS(2-60)treated group (FIG. 27D). Moreover, one mouse treated with Fc-HRS(2-60)showed Treg levels similar to naïve animals. Together, these resultssuggest that TNBS alters T cell populations in the spleen to a moreinflammatory phenotype through increased CD8⁺ T cells and decreased Tregcells and that treatment with Fc-HRS(2-60) restores these populationstowards homeostatic levels.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-86. (canceled)
 87. A method of reducing a lung inflammatory responsein a subject in need thereof, comprising administering to the subject acomposition comprising a histidyl-tRNA synthetase (HRS) fusionpolypeptide having at least one Fc region (HRS-Fc fusion polypeptide),wherein the HRS-Fc fusion polypeptide has an amino acid sequence that isat least 95% identical to SEQ ID NO:337, wherein the HRS-Fc fusionpolypeptide is at least about 95% pure and less than about 5%aggregated, and wherein the composition is substantially endotoxin-free.88. The method of claim 87, wherein the HRS-Fc fusion polypeptidereduces immune cell activation and invasion into damaged lung.
 89. Themethod of claim 87, wherein the HRS-Fc fusion polypeptide increasesproduction of anti-inflammatory cytokines, reduces production oractivity of pro-inflammatory cytokines, or both.
 90. The method of claim87, wherein the HRS fusion polypeptide comprises an amino acid sequenceat least 97% identical to SEQ ID NO:
 337. 91. The method of claim 90,wherein the HRS fusion polypeptide comprises an amino acid sequence atleast 98% identical to SEQ ID NO:
 337. 92. The method of claim 87,wherein the Fc region and the HRS polypeptide are separated by a peptidelinker.
 93. The method of claim 87, wherein the HRS-Fc fusionpolypeptide is substantially in dimeric form in a physiologicalsolution.
 94. The method of claim 87, wherein the HRS-Fc fusionpolypeptide has substantially the same secondary structure as acorresponding unmodified HRS polypeptide, as determined via UV circulardichroism analysis.
 95. The method of claim 87, wherein the HRS-Fcfusion polypeptide has a plasma or sera pharmacokinetic AUC profile atleast 5-fold greater than a corresponding, unmodified HRS polypeptide.96. The method of claim 87, comprising intravenously administering thecomposition to the subject.
 97. The method of any one of claims 87-96,wherein the subject has problems with breathing.
 98. The method of claim97, wherein the problems with breathing comprise shortness of breath, anon-productive cough, or both.